28 <strong>Bt</strong> <strong>Brinjal</strong>: <strong>The</strong> <strong>GEAC</strong> <strong>environmental</strong> <strong>risk</strong> <strong>assessment</strong>BOX 6Taxa known to form fertile hybrids with Solanum melongena L.<strong>The</strong> taxonomy <strong>of</strong> Solanum melongena L. <strong>and</strong> related species has changed over <strong>the</strong> years as modern taxonomic methods havebeen brought to bear. Consequently <strong>the</strong> status <strong>of</strong> published work documenting hybridization with S. melongena should bere-evaluated in light <strong>of</strong> <strong>the</strong>se more modern taxonomic concepts.1. <strong>The</strong> species concept <strong>of</strong> Solanum melongena L. now includes wild relatives that used to be considered separate species, <strong>the</strong>most notable <strong>of</strong> which was S. insanum, now S. melongena var. insanum. Cultivated varieties <strong>of</strong> S. melongena can crosswith S. melongena var. insanum (Ali <strong>and</strong> Fujiera 1989, Deb 1989, Lester <strong>and</strong> Hasan 1991a, Karihaloo <strong>and</strong> Gottlieb 1995,Karihaloo et al. 1995, Kashyap et al. 2003).2. Solanum incanum L. has been considered to be closely related to S. melongena L. Modern methods show it is a distincttaxon from S. melongena, <strong>and</strong> it may be closely related to S. melongena (Furini <strong>and</strong> Wunder 2004), or as distantly relatedas S. macrocarpon L. (Isshiki et al. 2008) or <strong>of</strong> intermediate status (Levin et al. 2006). Fertile hybrids <strong>of</strong> S. melongena <strong>and</strong>S. incanum occur (Deb 1989, Lester <strong>and</strong> Hasan 1991a, Karihaloo <strong>and</strong> Gottlieb 1995, Karihaloo et al. 1995, Behera <strong>and</strong>Singh 2002) <strong>and</strong> Viswanathan (1975) found a putative hybrid between S. melongena <strong>and</strong> S. incanum growing along aroadside in Kerala, India.3. Solanum violaceum Ortega has been considered to be closely related to S. melongena, but not as close a S. incanum.Solanum indicum L. has been synonymized to S. violaceum Ortega. Modern methods place it closer to S. melongena thanS. macrocarpon (Levin et al. 2006, Isshiki et al. 2008), but it may be a closer (Isshiki et al. 2008) or more distant (Levin etal. 2006) relative than S. incanum. Fertile hybrids are formed with S. melongena (Ali <strong>and</strong> Fujieda 1990, Patel et al 2001,Behera <strong>and</strong> Singh 2002 [as pollen parent]), although some hybrids are only partially fertile (Kashyap et al. 2003)4. Solanum macrocarpon L. has classically been included in <strong>the</strong> Macrocarpon Series or <strong>the</strong> S. macrocarpon species group(Whalen 1984), which is separate from <strong>the</strong> close relatives <strong>of</strong> S. melongena L. Modern taxonomic studies support thisseparation (Furini <strong>and</strong> Wunder 2004, Levin et al. 2006, Isshiki et al. 2008, Polignano et al. 2010). Fertile hybrids havebeen formed (Schaff et al. 1982), but not always (Gowda et al. 1990).5. Solanum aethiopicum L. has classically been included in <strong>the</strong> Anguivi Series or <strong>the</strong> S. anguivi species group (Whalen 1984)<strong>and</strong> modern analysis suggests that it is more closely allied with S. macrocarpon than S. melongena (Furini <strong>and</strong> Wunder2004, Levin et al. 2006). Fertile hybrids are produced (Ali <strong>and</strong> Fujieda 1990, Behera <strong>and</strong> Singh 2002 [as pollen parent]),but some hybrids are sterile <strong>and</strong> some are only partially fertile (Kashyap et al. 2003).6. Solanum virginianum L. has been considered more distant than any <strong>of</strong> <strong>the</strong> previously mentioned species. Modernmethods place it closer than S. incanum <strong>and</strong> S. macrocarpon (Isshiki et al. 2008) or more distant than all <strong>of</strong> <strong>the</strong> speciesdiscussed here (Levin et al. 2006). Partial fertility was established by Ali <strong>and</strong> Fujieda (1989), <strong>and</strong> Kashyap et al. (2003)report that hybrids are mostly fertile.7. Four additional species have been reported to form viable hybrids with S. melongena. <strong>The</strong>se are Solanum anomalumThonn., which forms viable hybrids as <strong>the</strong> seed parent (Behara <strong>and</strong> Singh 2002), Solanum gr<strong>and</strong>iflorum Ruiz & Pav.(Rameriz 1959), Solanum sodomeum (Tudor <strong>and</strong> Tomescu 1995), <strong>and</strong> Solanum undatum Lam. (Capinpin et al. 1963,Fukusawa 1964). <strong>The</strong> affinities <strong>of</strong> <strong>the</strong>se taxa with S. melongena has not been investigated with modern techniques. S.sodomeum has been classically grouped with S. virginianum in <strong>the</strong> Sodomela Series.incanum have already been addressed above, so <strong>the</strong> o<strong>the</strong>r four species are considered here. One <strong>of</strong> <strong>the</strong> species tested, S. nigrumis not in <strong>the</strong> subgenus Leptostemonum (SS 2010), <strong>and</strong> <strong>the</strong>refore cannot hybridise with brinjal. <strong>The</strong> remaining three speciesare in Leptostemonum. No viable hybrids were formed with any <strong>of</strong> <strong>the</strong>se species, S. violaceum, S. sisymbrifolium, <strong>and</strong> S.torvum. Solanum sisymbrifolium is a South American species that must have recently invaded India. Fertile hybrids betweenS. melongena <strong>and</strong> ei<strong>the</strong>r S. sisymbrifolium or S. torvum have never been reported, despite multiple attempts (Kashyap et al.
Environmental Risk Assessment 292003). IIVR (2007a) can be considered to confirm <strong>the</strong>se previously published results. However, multiple papers have reportedsuccessful hybridisation between brinjal <strong>and</strong> S. violaceum (Box 6). Consequently <strong>the</strong> results in IIVR (2007a) for S. violaceummust be considered anomalous. Taking all <strong>of</strong> <strong>the</strong>se issues into consideration, as a whole, <strong>the</strong> data in IIVR (2007a) are ra<strong>the</strong>runinformative for assessing <strong>the</strong> gene flow <strong>risk</strong>s associated with <strong>Bt</strong> brinjal.Nine species <strong>of</strong> Leptostemonum are known to hybridise with brinjal (Box 6), <strong>and</strong> six <strong>of</strong> <strong>the</strong>se species are known to occur inIndia. Twenty-six species <strong>of</strong> Leptostemonum occur in India, <strong>and</strong> <strong>the</strong> brinjal hybridisation potential for almost 20 <strong>of</strong> <strong>the</strong>se speciesis unknown. Only three <strong>of</strong> <strong>the</strong> 26 species were even considered by EC-II, <strong>the</strong> Dossier, or <strong>the</strong> Supplemental Materials. Thus<strong>the</strong>re was insufficient information for concluding that gene flow <strong>risk</strong>s are low.Needed Experiments <strong>and</strong> ObservationsAdditional analysis <strong>and</strong> experimentation should be conducted so that gene flow <strong>risk</strong>s to o<strong>the</strong>r species <strong>of</strong> Solanum insubgenus Leptostemonum can be adequately assessed (numbering is continued from previous section):2) Identification <strong>of</strong> wild relatives. Additional effort should go into determining if <strong>the</strong>re are more species <strong>of</strong> Leptostemonumthat occur in India. <strong>The</strong> nation’s herbaria should be examined <strong>and</strong> taxonomic experts in <strong>the</strong> group should be consulted.3) Hybridisation. <strong>The</strong> 26 species listed in Box 4, <strong>the</strong> three additional species listed in Box 6, <strong>and</strong> any new species identifiedin <strong>the</strong> previous efforts in 1) should be carefully analysed to determine which species are known not to hybridise with brinjal.4) Overlap. For <strong>the</strong> species that remain from 2), if <strong>the</strong> data exist, <strong>the</strong> timing <strong>of</strong> flowering <strong>and</strong> <strong>the</strong> geographic distributionshould be characterised to determine <strong>the</strong> extent <strong>of</strong> overlap with brinjal.5) Cross-compatibility. For species with overlap (<strong>and</strong> species for which <strong>the</strong> information is insufficient to conclude that <strong>the</strong>reis no overlap), cross-compatibility between <strong>the</strong> species <strong>and</strong> brinjal should be evaluated experimentally using methods that willprovide more accurate estimates <strong>of</strong> successful hybridisation. To include relevant genetic diversity, <strong>the</strong> hybridisation studiesshould use recently collected wild <strong>and</strong> weedy genotypes (not older accessions) from a range <strong>of</strong> habitats <strong>and</strong> geographic areas inIndia (e.g., see Karihaloo <strong>and</strong> Gottlieb 1995), especially regions where brinjal production is concentrated. <strong>The</strong>se experimentsshould be designed to confirm previously published results on successful <strong>and</strong> unsuccessful hybridisation so that <strong>the</strong> data on newspecies can be considered to be reliable. If <strong>the</strong> previous studies cannot be confirmed, it will be necessary to conduct additionalexperiments to explain why. By doing this, <strong>the</strong> data from <strong>the</strong> hybridisation experiments will be more reliable.6) Fitness <strong>of</strong> interspecific hybrids <strong>and</strong> backcrosses. For <strong>the</strong> species in Box 6 <strong>and</strong> any o<strong>the</strong>r species forming fertile hybridswith brinjal, experiments should be conducted to determine <strong>the</strong> relative fitness <strong>of</strong> crosses between <strong>the</strong> hybrids <strong>and</strong> <strong>the</strong> wildparent (this cross is called an F1 backcross). If <strong>the</strong> fitness <strong>of</strong> <strong>the</strong> backcross progeny is greater than <strong>the</strong> F1 hybrid, <strong>the</strong>n <strong>the</strong>species should be considered at <strong>risk</strong> <strong>of</strong> gene flow.7) Gene flow rates. It will probably be a waste <strong>of</strong> time to conduct additional field experiments to estimate gene flowrates from <strong>Bt</strong> brinjal to <strong>the</strong> wild species. It is difficult to obtain unbiased estimates <strong>of</strong> gene flow, <strong>and</strong> for <strong>the</strong> purpose <strong>of</strong> <strong>risk</strong><strong>assessment</strong> it can be assumed that gene flow is high enough to be evolutionarily <strong>and</strong> ecologically meaningful.Finding 12. <strong>The</strong>re is insufficient evidence that wild or weedy relatives <strong>of</strong> brinjal would not obtain a fitness benefitfrom a <strong>Bt</strong> transgene should gene flow occur. <strong>The</strong>re is insufficient evidence that wild relatives <strong>of</strong> brinjal will not sufferreduced genetic diversity from <strong>the</strong> introgression <strong>of</strong> <strong>the</strong> <strong>Bt</strong> transgene. <strong>The</strong>re is insufficient evidence that non-GM brinjalwill remain uncontaminated by <strong>Bt</strong> brinjal.EC-II (page 56, our emphasis) asserts “FSB is a lepidopteran pest that prefers only brinjal <strong>and</strong> cry1Ac provides protectiononly against FSB <strong>and</strong> o<strong>the</strong>r lepidopteran pests. Since no lepidopteran pests are prevalent on Solanum wild species, <strong>the</strong> matter <strong>of</strong>fitness advantage does not arise.” No evidence is provided to substantiate <strong>the</strong>se claims. <strong>The</strong>re are no published studies on <strong>the</strong>arthropod herbivores (including lepidoptera) inhabiting wild populations <strong>of</strong> S. melongena, S. melongena insanum, or S. incanumor any <strong>of</strong> <strong>the</strong> o<strong>the</strong>r wild relatives potentially at <strong>risk</strong> (Box 4 <strong>and</strong> Box 6). Thus, it is possible that some wild relatives <strong>of</strong> brinjalwould gain an advantage from <strong>the</strong> <strong>Bt</strong> transgene.<strong>The</strong> host range <strong>of</strong> BFSB is uncertain. Even if EC-II is correct in that BFSB prefers to feed on brinjal, it is <strong>the</strong> suitablehost range, not <strong>the</strong> preferred host range that is relevant for assessing this <strong>risk</strong>. All things being equal, <strong>the</strong> preferred host will