Plant Science 170 (2006) 953–961 www.elsevier.com/locate/plantsci Infertile interspecific hybrids between transgenically mitigated Nicotiana tabacum and Nicotiana sylvestris did not backcross to N. sylvestris Hani Al-Ahmad a, Shmuel Galili b, Jonathan Gressel a,* a Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel b Agronomy and Natural Resources Department, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel Received 28 August 2005; received in revised form 24 December 2005; accepted 3 January 2006 Available online 2 February 2006 Abstract Genes may introgress from transgenic crops into sexually compatible wild relatives via pollen flow. This could potentially enhance the ecological expansion of the introgressed hybrids and their progeny at the cost of other plant species, or affect health of humans and animals, depending on the novel trait engineered into the crop. To prevent generating such competitive transgenic progeny, we previously used tobacco (Nicotiana tabacum L.) as a model for validating a transgenic mitigation (TM) mechanism using tandem constructs where a gene of choice is linked to mitigating genes that are positive or neutral to the crop, but deleterious to a recipient when in competition with the wild type. In the present study, attempts were made to achieve interspecific sexual hybridization between transgenic TM allotetraploid N. tabacum (pollen donor, representing a crop bearing novel traits) into one of its progenitors, diploid wild type Nicotiana sylvestris (representing a wild relative as well as a progenitor). N. sylvestris plants were manually pollinated by transgenic tobacco. The F1 interspecific sexual hybrids had >75% pollen sterility and produced no seeds. When the F1 was backcrossed as the pollen donor to N. sylvestris, the progeny produced almost no germinable seeds. With such low risk of gene flow, transgenic tobacco bearing novel traits could be cultivated with minimal concern where N. sylvestris is a native or ornamental species. # 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Gene flow; Infertility; Interspecific sexual hybridization; Nicotiana tabacum; Nicotiana sylvestris; Transgenic mitigation 1. Introduction weedy relatives, and the potential impact this may have on the environment and on toxicity to other organisms. Transgenic crops can interbreed with related wild or weedy Tobacco (Nicotiana tabacum L.) is commercially cultivated species by hybridization and backcrossing, potentially enhan- for its leaves and is one of the best model species used in cing the invasiveness in the progeny [1–4]. Some of the transgenic research as it can easily be genetically transformed engineered genes such as those conferring resistance to and regenerated. It is widely used to test suitability of plant- herbicides, pathogens and to environmental stresses could based systems for bio-production of recombinant proteins [5].It provide a fitness advantage to a wild or weedy species, would be necessary to consider cultivating transgenic tobacco especially when the crop and its relative species grow in the plants in containment systems if genetically related wild or same agroecosystem. There is also concern about transgene weedy Nicotiana species were present that might introgress flow from crops bearing pharmaceutical or novel industrial transgenes from engineered tobacco plants. traits into other varieties of the same species or into wild or The genus Nicotiana contains 77 diploid and polyploid species [6], updated from 65 taxonomically characterized species by Ref. [7]. N. tabacum is a natural allotetraploid Abbreviations: TM, transgenic mitigation/transgenically mitigated (2n =4x = 48) species. Based on flower morphology, chromo- * Corresponding author. Tel.: +972 8 934 3481; fax: +972 8 934 4181. some segregation patterns, chloroplast and mitochondrial E-mail address: [email protected] (J. Gressel). sequence data, N. tabacum is thought to be derived from 0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2006.01.005 954 H. Al-Ahmad et al. / Plant Science 170 (2006) 953–961 two presumed progenitors [7–11]: Nicotiana sylvestris (diploid, Various methods have been proposed to limit gene flow by: 2n =2x = 24) is the closest extant species with a similar S- (1) ‘‘containment’’, i.e. to keep the gene from appearing in genome donor, and based on plastome and chondriome hybrids by using male sterility, gene targeting to the plastome, homology, it is thought to be the maternal parent [12,13], genetic use of restriction technologies (GURT), but all of and; either of two very similar species Nicotiana tomentosi- these are either uni-direction preventing outflow or influx, or formis and Nicotiana otophora (diploids, 2n =2x = 24). These are leaky [4,26] and (2) ‘‘transgenic mitigation (TM)’’ where shrubby species, with a limited range in Bolivia–Argentine the transgene of choice is tandemly linked to mitigating border regions, are thought to be the most likely paternal T- genes that are positive or neutral to the crop, but deleterious to a genome donor as they share extensive sequence homology to recipient under competition [26,27]. Such mitigator genes the N. tabacum T-genome [8]. Currently, N. tomentosiformis is render the relatives unfit to compete, keeping individuals favored as the T-genome progenitor because its chromosomes bearing TM traits at very low frequencies due to competition. pair better with N. tabacum than N. otophora [9–11,14]. N. Dwarfing, anti-seed shattering and anti-secondary dormancy sylvestris is an annual to perennial herb, originating from should be appropriate mitigator genes for many row crops. Argentine/Bolivia, the same area where N. tabacum probably Special mitigator genes are available for special crops, e.g. originated [7]. N. tabacum has long been grown in many anti-bolting for biennials, enhanced root growth for temperate to subtropical regions [7], and today it is phytoremediating species or shrunken kernels for pharma- commercially cultivated for tobacco products in most parts ceutical species. We have previously demonstrated the of the world. N. sylvestris is widely cultivated in gardens around effectiveness of the mitigation concept using N. tabacum as the world as an ornamental species. a model, with herbicide resistance as the primary gene and Interspecific sexual hybridization is often prevented by dwarfing as the mitigator. We showed that the Dgai-gene incompatibility barriers that are activated at different stages of expressing dwarf plants were highly productive when grown growth, but there are fewer barriers to crossing with progenitors alone, but weak and too unfit to reproduce when grown because of the relatedness. Both pre- and post-zygotic barriers interspersed at various proportions with the wild type [28,29] have the potential to reduce gene flow between species, e.g. at distances typical for weeds. Mitigation reduced the risk of different flowering times, gametic and zygotic incompatibility, transgene spread to other non-transgenic N. tabacum cultivars lack of hybrid viability and hybrid sterility [15]. We chose to andtorelatedNicotiana wild or weedy species grown in the ascertain the possibilities of hybridization and backcrossing area, as well. We also showed that in the particular case of with N. sylvestris because of its relatedness as the putative tobacco, intraspecific Dgai-hybrids with non-transgenic plants female progenitor. It could be expected to be the most likely were male sterile [28]. As the male sterility could be reversed species to introgress tobacco transgenes as it is widely by kinetin, Dgai could serve as both a gene for containment as cultivated as an ornamental species. The other putative well as a gene for mitigation [30]. We also demonstrated progenitors were not tested as they are highly localized [7], that mitigation with the same gene construct could be useful in and the ambiguity of paternity [9]. prevent oilseed rape (Brassica napus) from establishing If compatibility barriers are incomplete, hybrids may be as a volunteer weed in the following crop [31],orinhybrids formed and function as a bridge forgene transfer. No publications with and backcrosses to weedy Brassica rapa = Brassica could be found on normal sexual hybridization between N. campestris [32]. tabacum and N. sylvestris with fertile progeny. The only way that In this study, we attempted to demonstrate transgene flow fertile progeny could be obtained was by doubling the and introgression from N. tabacum (as a crop) into N. sylvestris chromosomes of the F1 by colchicine treatment [16]. Using (as a wild relative and as a progenitor). Crossing of crops with such techniques to move genes is arduous for the researcher, and their progenitors or with weeds derived from the same is unlikely to occur successfully in nature. Although, breeders progenitor or similar to the crop has been known to create have moved valuable genes from wild Nicotiana species into worse weed problems than before, as has been evidenced with cultivated tobacco [17,18], they would not have tried to beet [33], foxtail millet (Setaria italica) [34] and rice, e.g. Ref. backcross crop genes into the wild or weedy species, as gene [35]. We report that the F1 interspecific sexual hybrids between flow to wild or weedy species was hardly of interest until the N. sylvestris and N. tabacum were infertile, and failed to advent of transgenics and issues related to gene flow to the wild introgress the transgenes in