Laris s a A Guimarães 4, Andrea D Koehler

1_{Embrapa Recursos Genéticos e Biotecnologia Brasília – DF, Brazil.} 2_{Embrapa Gado de Corte, Campo Grande, MS, Brazil.}

3_{Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ. Brazil.}

4_{Universidade de Brasília, Brasília, DF. Brazil.} 5_{CENA, Piracicaba, São Paulo, SP, Brazil.}

In Brazil most of the cattle is raised under pasture conditions with around 196 million heads distributed in an area of approximately 259 million hectares, 115 million covered mostly by two apomictic cultivars of

to this low genetic variability, there is an important concern on these cultures mainly because of apomixis. Therefore, studies on the molecular mechanisms involved on the reproductive mode of Brachiaria aiming the knowledge of apomixis is conducted by our group. Genetic studies

suggested that apomixis is a dominant factor controlled by one Mendelian locus. Apomixis is of the aposporic type, pollen is viable and pseudogamy occurs, i.e. endosperm is a triploid tissue resulting from the fertilization of the central cell while the development of the embryo is autonomous. Comparing to apomictic plants, the seed production of sexual plants of B.

brizantha is limited by a massive abortion of developing caryopsis, most

likely associated with early inbreeding depression. At the molecular level, cDNA sequences from ovaries show differential expression in sexual and apomictic plants and among them, some have evident expression in the synergids, which could be involved with the fertilization and autonomous development of unreduced egg cell. To test the function of candidate genes for apomixis, in vitro regeneration methods using meristem and cell suspension of Brachiaria were established allowing the improvement of transformation protocols by biolistics. Advances towards obtaining new intraspecific lines were achieved with evidence of compatibility of B.

brizantha induced tetraploid sexual and natural tetraploid, apomictic plants.

A segregant population of B.humidicola for apomixis was recently obtained and RAPD analysis is being performed, to confirm hybrids and genetic mapping is on the way with candidate genes for apomixis of other species and SSR markers.

92

Containment of trans genes by maternal inheritance or

cytoplas mic male s terility engineered via the chloroplas t genome

Henry Daniell

Department of Molecular Biology and Microbiology, University of Central Florida, Orlando, FL 32816-2364, U. S. A. daniell@mail.ucf.edu

Maternal inheritance or cytoplasmic male sterility is a promising approach for transgene containment via chloroplast genetic engineering, with added advantages of high levels of transgene expression (up to 47% of total leaf protein), rapid multigene engineering, lack of position effect, gene silencing and pleiotropic effects. Currently, chloroplast genetic engineering has been utilized widely in tobacco, a non-food/feed crop as a bioreactor for production of amino acids, biopharmaceuticals, biopolymers or industrial enzymes. Several other crop chloroplast genomes have been transformed via organogenesis (cauliflower, cabbage, lettuce, oilseed rape, petunia, poplar, potato, tomato) or embryogenesis (carrot, cotton, rice, soybean) and maternal inheritance of transgenes have been observed.

Several crop species have stably integrated transgenes conferring agronomic traits including herbicide, insect and disease resistance, drought and salt tolerance, and phytoremediation. Chloroplast transgenic carrot plants

withstand salt concentrations that only halophytes could tolerate. Insecticidal proteins engineered via the chloroplast genome killed insects that had

developed 40,000 – fold resistance against that protein. Forty sequenced crop chloroplast genomes provide information on plastid genome regulatory sequences and spacer regions for transgene integration.

Chloroplast-derived biopharmaceutical proteins including insulin, interferons and somatotropin have been evaluated by in vitro studies. Human interferon alpha 2B transplastomic plants have been evaluated in field studies. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, Lyme disease), viral (canine parvovirus, rotavirus, etc) and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies and pathogen or toxin challenge in animals. Oral delivery of proinsulin offered protection against development of insulitis (diabetes) in mice; such delivery eliminates expensive fermentation,

purification, low temperature storage and transportation. Demonstration of these biotechnology applications bode well for commercial development utilizing this new platform technology.

Evaluation of an apomictic genotype of B rachiaria brizantha

leading to cultivar releas e and protection

Cacilda B orges do Valle1*_{, Valéria Pacheco B atista Euclides}1*_{, José Raul}

Valério1_{, Manuel Claudio Motta Macedo} 1_{, Lucimara Chiari}1_{, Maria S uely}

Pagliarini2*_{, Liana Jank} 1*_{, Rosangela Maria S imeão Resende} 1_{, Moacyr}

B ernardino Dias -Filho3

1_{Embrapa Beef Cattle, Caixa Postal 154, 79002-970 Campo Grande, MS, Brazil} 2 _{Department of Cell Biology and Genetics, State University of Maringá, 87020-900}

Maringá PR Brazil.

3_{Embrapa Eastern Amazon, Caixa Postal 48, 66017-970, Belém, PA, Brazil.} *_{CNPq Scholars.}

The evaluation process leading to cultivar development of a forage grass is a long-term investment, which requires a multidisciplinary team. Forage plants are only valuable when transformed into high quality protein such as milk, meat, leather or hide. Therefore indirect measures of quantity and quality of the forage need to be undertaken starting with plot evaluation under a cutting regime all the way to animal performance trials, in pastures, under grazing. Not before 8-10 years is needed to confirm the usefulness and advantages of the candidate genotype. Apomixis adds an interesting aspect to forage cultivar development for at one hand, it simplifies seed multiplication and yields uniform pastures which are easier to manage, but on the other hand, impairs recombination of useful traits by hybridization if a sexual compatible genotype is not available. Apomixis is never obligate in most useful forage species, therefore there is always the possibility of the residual sexuality yielding some hybrid genotypes in the progeny.

Brachiaria reproduces predominantly by apomixis of the Panicum type and

is pseudogamous, thus the embryo is always a hybrid tissue. The path to cultivar development of BRS Piatã, the first Brachiaria cultivar protected by Embrapa, started in 1988 by agronomic trials in plots, established by cuttings since seed was not available and mode of reproduction had not been established at the time. Seed multiplication followed so that regional trial could be carried out between 1994 and 1997. Animal trials to study the effect of the animal on the pasture were started in 1997, in 1000 m2

paddocks replicated twice. To determine animal performance on BRS Piatã, 2-hectare pastures in two replicates were established and measurements were taken for three years. For over 10 years breeder’s seed was multiplied from the most vigorous plants in four different areas, and later, distributed for basic seed production. Twenty years of evaluation and selection were enough to produce differences picked up with molecular markers, between the original plants and those derived from breeder’s certified seed. Possible inferences will be discussed at the conference.

94

Identification of over expres s ed and down-regulated genes during

arabidops is meios is and micros porogenes is

Libeau P.1_{, Durandet M.} 1_{, Marquis C.} 1_{, Taconnat L.} 2_{, Renou J.P.} 2_,

Jenczewski E.1_{, Grelon M.}1_{, Mercier R.}1_{, Mezard C.}1_{, and Horlow C.}1

1_{INRA – Institut Jean-Pierre Bourgin – Station de Génétique et d’Amélioration des}

Plantes, Route de St Cyr – 78026 Versailles Cedex – France.

2_{URGV -UMR INRA 1165 – CNRS 8114 – UEVE – 2, rue Gaston Crémieux, CP5708,}

91057 Evry cedex – France.

In Eukaryotes, meiosis is a key function of sexual reproduction, a complex and specialized process of cell division that results in haploid cells (e.g; gametes).

In recent years, the availability of Arabidopsis genome sequence, the development of cytological approaches for this species and the combination of forward and reverse genetic approaches have allowed an important progress in the identification of meiotic genes in plants.

Nevertheless identification of a larger number of meiotic genes remains a challenge due to problems of sequence conservation among species and functional redundancy between gene family members.

Hence we have developed a transcriptomic-based approach comparing isolated Arabidopsis male meiocyte mRNAs to different plant organs to enable the identification of a greater range of plant genes involved in meiosis and microsporogenesis.

From transcriptome data, only 2% of total gene number (22144) are significantly expressed and 0.1% are down-regulated during meiosis pathway. Using Arabidopsis T-DNA knock-out mutants, we will confirm the role of candidate genes for meiosis and microsporogenesis in plants.