RESUMO
The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.
Assuntos
Oryza/genética , Plantas Geneticamente Modificadas/genética , Agrobacterium/fisiologia , Vetores Genéticos/genética , Oryza/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Transformação Genética/genéticaRESUMO
BACKGROUND AND AIMS: Cassava (Manihot esculenta), a major food staple in the tropics and subtropics, thrives even in environments undergoing threatening climate change. To satisfy the increasing demand for crop improvement and overcome the limitations of conventional breeding, the introduction of inbreeding techniques such as the production of doubled haploid lines via androgenesis or gynogenesis offers advantages. However, comprehensive studies on cassava flower bud biology or structural development are lacking and precise structural and biological information is a prerequisite to enhance the efficiency of these techniques. METHODOLOGY: The floral biology of three selected cassava lines was studied, focusing on morphology, phenology and pollen biology (quantity, viability and dimorphism). Histological studies were also conducted on microsporogenesis/microgametogenesis and megasporogenesis/megagameto-genesis to generate precise developmental data for these lines. PRINCIPAL RESULTS: Male and female cyathia have distinct developmental phases. Pollen viability was high during immature stages of plant development; however, pollen mortality was common at later stages. Pollen trimorphism in male gametophytes towards the larger or smaller pollen size, as compared with normal size, was observed. Ten characteristic events were identified in male gametogenesis and six in female gametogenesis that were correlated with flower bud diameter. Male gametophyte diameter at different developmental stages was also determined. CONCLUSIONS: Results indicate that the three lines did not differ significantly, except regarding a few morphological aspects such as plant height, flower colour and number of male cyathia. Pollen grains were initially viable, but viability decreased drastically at later stages of growth. Abnormal meiosis or mitosis triggered pollen trimorphism. The demonstrated sequential events of reproductive development generated valuable information at the cellular level, which will help close the current information gap for cassava improvement via breeding programmes and doubled haploid plant production.