Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.

Cryptic sexual populations account for genetic diversity and ecological success in a widely distributed, asexual fungus-growing ant

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Poliembrionia e aspectos da embriogênese em Tabebuia ochracea (Chamisso) Standley (Bignoniaceae)

A origem dos embriões supranumerários e a embriogenia de Tabebuia ochracea foram analisadas. Embriões supranumerários apomíticos têm origem adventícia a partir de células da hipóstase e do tegumento da região micropilar do óvulo. A embriogenia corresponde ao tipo Onagrado. Das 233 sementes dissecadas 81,37% apresentaram poliembrionia e foram encontrados até sete embriões em uma mesma semente. Aparentemente, embriões sexuais e adventícios podem se desenvolver juntos, numa mesma semente. Alguns dos embriões adventícios apresentam alterações morfoanatômicas graves que podem prejudicar seu desenvolvimento em plântulas.

Polyembryony increases embryo and seedling mortality but also enhances seed individual survival in Handroanthus species (Bignoniaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cytogenetics and embryology of Eupatorium laevigatum (Compositae)

Embryological studies indicate Eupatorium laevigatum to have Antennaria type diplospory with precocious embryony. The embryo sac is of the Polygonum type and the polar nuclei fuse before anthesis (maturation of the stamens). Endosperm development is autonomous and the central cell divides only after the initial stages of embryo formation. It is estimated that about 10% of the florets in anthesis contain an undivided egg which can be used for sexual reproduction. The study of microsporogenesis revealed abnormalities in chromosome pairing which result in the formation of univalents, bivalents, trivalents and higher polyvalents, with the consequent production of lagging chromosomes, unbalanced nuclei, micronuclei and sterile pollen. We found that, as represented by the material studied, E. laevigatum is an autohexaploid (2n = 6x = 60) in which each chromosome of a basic set of ten chromosomes is repeated six times and that E. laevigatum is an essentialy obligate apomictic.

Expressão protéica diferencial entre plântulas apomíticas e zigóticas de citros

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

RAPID analysis reveals genetic variability among sexual and apomictic Paspalum dilatatum poiret biotypes

Paspalum dilatatum is a valuable forage grass in the subtropics. This species consists of several sexual (tetraploid) and apomict (penta- and hexaploid) biotypes. It has been proposed that the presence of a genome of unknown origin, the X genome, is responsible for apomixis in penta- and hexaploid biotypes. Here we evaluated the utility of random amplified polymorphic DNA (RAPD) markers for discriminating sexual and apomictic P dilatatum biotypes. DNA samples from nine accessions, including P. intermedium, P. juergensh, and P dilatatum (ssp. flavescens, and the common and Uruguayan biotypes) were analyzed with 86 RAPID primers. Three hundred sixty-two fragments were scored and genetic similarity estimates revealed that the penta- and hexaploid biotypes were highly similar (S,, greater than or equal to 0.913). Forty RAPDs were unique to the penta- and hexaploid biotypes. Overall RAPID markers were useful for assessing genetic variation among closely related P dilatatum genotypes as well as generating putative X genome markers.

Breeding systems of shrubs and trees of a Brazilian savanna

The reproductive biology of shrubs and trees of a preserved savanna (''cerrado'') area in the municipality of Corumbatai São Paulo State, Brazil was studied. The floral sexuality of 135 species were characterized, with 85.2 % hermaphroditic, 9.4 % dioecious, 4.5 % monoecious, and one determine the breeding systems. Nine apomictic species were found, all belonging Melastomataceae. Among the twelve sexual reproducing species, seven (58.3 %) proved to be self-compatible, and five (41.7 %) self-incompatible. Anemophily was found in five species, although pollinations systems were not investigated in other species.

Identification and analysis of single nucleotide polymorphisms (SNPs) in citrus

Most of the cultivated species of citrus have narrow genetic basis. Relationships among species and cultivars are obscured by sexual compatibility, polyembryony, apomixis and a high incidence of somatic mutations. DNA analysis is crucial in genetic studies not only for citrus breeding programs but also for characterization of hybrids and species. In this paper, single nucleotide polymorphisms ( SNPs) were investigated in 58 accessions of Citrus, hybrids and related genera. Genomic sequences of 'Pera IAC' sweet orange ( Citrus sinensis L. Osbeck) were used for primer design and selection of sequence tagged sites (STSs) for identification of SNPs. Analysis of 36 STSs showed identical sequences among 40 of the 41 sweet orange accessions studied. However, these accessions were heterozygous for many SNPs. Ten selected STSs were analyzed in 17 additional accessions from 13 species and hybrids. Comparing to the 'Pera IAC' sweet orange accession, a total of 150 polymorphic nucleotides were identified and most of the alterations were transitions ( 52.7%). The greatest number of SNPs was observed in Poncirus trifoliata ( L.) Raf. and the smallest in 'Ponkan' mandarin ( Citrus reticulata Blanco). At the intra-specific level, 'Bafa Gigante' ( Citrus sinensis L. Osbeck) was the only sweet orange accession with a divergent SNPs genotype, which corroborates the hypothesis of a hybrid origin for this accession. Although the STSs analyzed represent randomly sampled genomic sequences, they provided consistent information about the level of polymorphism and showed the potential of SNPs markers for characterization and phylogenetic studies.

Self-fertility and polyembryony in South American yellow trumpet trees (Handroanthus chrysotrichus and H. ochraceus, Bignoniaceae): a histological study of postpollination events

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The reproductive biology of Cybistax antisyphilitica (Bignoniaceae), a characteristic tree of the South American savannah-like "Cerrado" vegetation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Karyotype Conservation in 2 Populations of the Parthenogenetic Scorpion Tityus serrulatus (Buthidae): rDNA and Its Associated Heterochromatin Are Concentrated on Only One Chromosome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Formation of multiple or adventive embryos in Epidendrum nocturnum Jacq (Orchidaceae)

Under greenhouse conditions, Epidendrum nocturnum Jacq. plants produce fruits by both self-fertilization and cleistogamy. Although adapted to these reproductive processes the species respond also to cross-pollination. Seeds without embryos and with one embryo are usual but occasionally seeds with two, three or four embryos are produced. Multiple embryos are formed by polyembryony and apomixis. © 1985 Annals of Botany Company.

Mating in the pseudogamic apomictic Anemopaegma acutifolium DC: Another case of pseudo-self-compatibility in Bignoniaceae?

Self-compatibility in apomictic pseudogamic species is considered fundamental to assure reproduction by seeds in extreme situations, making apomictic species more advantageous than sexual ones in these scenarios. Anemopaegma acutifolium is a polyploidy, apomictic sporophytic species with no endosperm development in ovules of unpollinated pistils, which indicates obligate pseudogamy. Thus, the aim of the present work is to study the breeding system and post-pollination events to test if there is similar pseudogamous development irrespective of pollination treatment. We analysed fruit and seed set obtained in controlled experimental pollinations, as well as embryo number per seed, and the progress of ovule penetration, fertilisation and early endosperm development between self- and cross-pollinated pistils. We found that the species is self-fertile and that spontaneous selfing fruit set is also possible, although emasculated flowers never form fruits. Selfed pistils were as efficient as crossed ones for all parameters analysed, except for a delay in endosperm development observed in the former that may be an effect of the late-acting self-incompatibility. Therefore, the avoidance of selfed pistil abortion seems to be promoted by the presence of adventitious embryos and a normal endosperm. We conclude that A. acutifolium shows apomixis-related pseudo-self-compatibility, as in other self-fertile apomictic species of Bignoniaceae, which confer reproductive assurance and increases fruit-set and persistence ability in fast-changing tropical habitats. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.