Histology of somatic embryogenesis in rice (Oryza sativa cv. 5272) / Histología de la embriogénesis somática en arroz (Oryza sativa cv. 5272)

Rice (Oryza sativa cv. 5272) embryogenic calli were obtained from mature zygotic embryos culture on Murashige & Skoog (1962) medium supplemented with 2.5 mg/l 2,4- dichlorophenoxyacetic acid. Histological analysis of somatic embryogenesis revealed that after two weeks of culture of explants on the callus induction medium, somatic embryo development began with a cluster of proembryogenic cells in the peripheral region of the calli. The outer cell layer of embryogenic calli consisted of small and isodiametric cells with a dense cytoplasm and a prominent nucleus and nucleolus; whereas the inner cell layer is composed of large cells with small nucleus and large vacuole. These embryogenic cells underwent a series of organized divisions and formed the proembryo with a well-defined protodermis. Rev. Biol. Trop. 57 (Suppl. 1): 141-150. Epub 2009 November 30.

Los estudios anatómicos e histológicos de los callos embriogénicos de arroz (Oryza sativa) mostraron que estos se originan del epitelio escutelar de los embriones cigóticos maduros. Al crecer en un medio Murashige y Skoog (1962) suplementado con 2.5 mg/l 2,4-D, presentan grupos de células embriogénicas en las zonas periféricas, las cuales a su vez darán lugar a la formación de proembriones y de embriones somáticos. Las células de los callos embriogénicos se caracterizan por tener núcleo y nucleolo conspícuos, forma isodiámetrica, citoplasma denso y están acompañadas de células adyacentes con abundantes gránulos de almidón. Los embriones somáticos completan su desarrollo para dar formación a plántulas completas, al estar en un medio de regeneración. Los estudios histológicos permitieron observar la expresión transitoria del gen uidA en grupos de células de las capas más externas de los callos embriogénicos sometidos al método de transformación genética por biobalística.

Elevada diversidade genética interpopulacional em Oryza glumaepatula Steud. (Poaceae) avaliada com microssatélites / High interpopulational genetic diversity of Oryza glumaepatula Steud. (Poaceae) assessed with microsatellites

Marcadores microssatélites foram usados para caracterizar a diversidade genética entre e dentro de sete populações naturais de Oryza glumaepaula. Seis dessas populações são originárias da bacia hidrográfica da Amazônia e uma do rio Paraguai no Pantanal Matogrossense. Utilizando sete locos de microssatélites, observou-se diversidade genética intrapopulacional média de 1,98 alelos por loco, 56,2 por cento de locos polimórficos, Ho = 0,026 e He = 0,241. Elevada diferenciação interpopulacional foi observada pelo índice de fixação de Wright e pelo parâmetro de divergência de Slatkin (F ST = 0,715 e R ST = 0,595, respectivamente), bem como elevado nível de endogamia total (F IT = 0,963), em grande parte influenciada pelo sistema reprodutivo (F IS = 0,858). Verificou-se que nenhuma população estava em equilíbrio de Hardy-Weinberg, devido ao predomínio da autofertilização, o que também pôde ser verificado pela taxa média aparente de cruzamentos: t a = 0,055. Consequentemente, o fluxo gênico entre populações foi praticamente nulo o que contribuiu para o elevado nível de divergência interpopulacional. De modo geral, as taxas de cruzamento foram muito baixas ou nulas nas populações da Amazônia. Entretanto, a população PG-3 do Rio Paraguai, originária do Pantanal Matogrossense, apresentou taxa de cruzamento mais elevada (13,2 por cento), indicando sistema reprodutivo misto com predomínio de autogamia. Como a diversidade intrapopulacional foi baixa, os resultados indicam que a amostragem de elevado número de populações é a estratégia mais adequada para a conservação ex situ desta espécie. Para a conservação in situ, com base na riqueza alélica, recomenda-se como prioridade as populações PG-3, TA-3, SO-17 e NE-7, originárias das bacias hidrográficas dos Rios Paraguai, Tapajós, Solimões e Negro, respectivamente.

Microsatellite markers were used to characterize the genetic diversity within and among seven natural populations of Oryza glumaepaula. Six of these populations originated from the hydrographic basin of the Amazon and one from Rio Paraguay in the Pantanal Matogrossense. Using seven microsatellite loci, the following intrapopulation genetic diversity parameters were estimated on average: 1.98 alleles per locus, 56.2 percent polymorphic loci, Ho = 0.026 and He = 0.241. High interpopulational differentiation was noticed by examining Wright's fixation index and Slatkin's divergence parameter (F ST = 0.715 and R ST = 0.595, respectively), as well as a high level of total inbreeding (F IT = 0.963), greatly influenced by the mating system (F IS = 0.858). No population was in Hardy-Weinberg equilibrium, due to the prevailing autogamic mating behavior, as also indicated by the average apparent outcrossing rate observed: t a = 0.055. Consequently, among populations gene flow was practically absent, which has contributed to the high interpopulational genetic divergence. In general, very low or null outcrossing rates were found in the Amazonian populations. However, population PG-3 from Rio Paraguay, originated from Pantanal Matogrossense, showed a higher outcrossing rate (13.2 percent), indicating a mixed mating system with the predominance of self-fertilization. Since intrapopulation diversity was low, results indicate that sampling a large number of populations is the most appropriate strategy for the ex situ conservation of this species. For in situ conservation, taking in consideration the allelic richness, the following populations are indicated as priority: PG-3, TA-3, SO-17, and NE-7, from the hydrographic basins of the rivers Paraguay, Tapajos, Solimoes and Negro, respectively.

Effect of 2,4-D, hydric stress and light on indica rice (Oryza sativa) somatic embryogenesis

With the purpose of increasing the embryogenesis regeneration process in vitroplants obtained from somatic embryos of the indica rice variety CR-5272 (Oryza sativa L.), two independent experiments were performed. The first experiment consisted in the effect of combination of three concentrations of the gelling agent PhytagelTM (1.8, 2.4, and 3 gL-1) and four 2,4-D concentrations (2.26, 4.52, 6.78, and 9.05 m M) on the induction and subsequent regeneration of embryogenic calli. On the second experiment, the pre-regeneration phase was modified; calli were subjected to darkness or diffuse light conditions for one, two, and three weeks. In embryogenesis induction, 35% calligenesis was obtained using the MS culture medium supplemented with 6.78 m M of 2,4-D and 2.4 gL-1 PhytagelTM , whereas on the control treatment (MS medium supplemented with 9.05 m M of 2,4-D and 3 gL-1 PhytagelTM ) 24% calligenesis was obtained. In addition, regeneration percentages were improved (22% and 16% for calli induced with the above treatments, respectively). Furthermore, in light exposure experiments, the best result was obtained by exposing the embryogenic calli to darkness for one week in pre-regeneration, followed by direct light exposure during the regeneration phase

Plant regeneration of indica rice (Oryza sativa) cultivars from mature embryo-derived calli

Plant regeneration from seven-week-old callus cultures derived from mature embryos of several indica rice cultivars was achieved with frequencies of morphogenic calli from 10 to 47 percent. Three media were tested both for callogenesis and plant regeneration. For 3 of the 7 genotypes examined, the best combination of media for plant regeneration was Murashige & Skoog basal medium: MSC (callogenesis) and MSR (regeneration). The rates of callogenesis were not related to the capacity for plant regeneration. Two genotypes CR-1113 and CR-5272 produced the highest number of regenerated green plants. The results of this study suggest that genetic differences could be directly linked to the ability to regenerate in these plant cultivars