In close agreement with visible germination, orthodox seeds lose desiccation tolerance (DT). This trait can be regained under osmotic stress, but the mechanisms are poorly understood. In this study, germinating seeds of Caragana korshinskii Kom. were investigated, focusing on the potential modulating roles of reactive oxygen species (ROS) in the re-establishment of DT. Germinating seeds with 2 mm long radicles can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution (-1.7 MPa). Upon PEG incubation, ROS accumulation was detected in the radicles tip by nitroblue tetrazolium chloride staining and further confirmed by confocal microscopy. The PEG-induced re-establishment of DT was repressed when ROS scavengers were added to the PEG solution. Moreover, ROS act downstream of abscisic acid (ABA) to modulate PEG-mediated re-establishment of DT and serve as a new inducer to re-establish DT. Transcriptomic analysis revealed that re-establishment of DT by ROS involves the up-regulation of key genes in the phenylpropanoid-flavonoid pathway, and total flavonoid content and key enzyme activity increased after ROS treatment. Furthermore, DT was repressed by an inhibitor of phenylalanine ammonia lyase. Our data suggest that ROS play a key role in the re-establishment of DT by regulating stress-related genes and the phenylpropanoid-flavonoid pathway.

Desiccation tolerance (DT) is the crucial ability of seeds to resist desiccation. However, the regulatory mechanisms of seed DT are not fully understood. In this study, two heat shock cis-elements (HSEs) were identified in the Brassica napus galactinol synthase (BnGolS1) promoter and shown to bind the heat shock transcription factor A4a (BnHSFA4a). Transcriptional expression of BnHSFA4a was induced at the early stage of DT acquisition, prior to increased BnGolS1 activity and galactinol production. Ectopic overexpression of BnHSFA4a (oxBnHSFA4a) in Arabidopsis enhanced DT, particularly during DT re-establishment. OxBnHSFA4a up-regulated the expression of GolS1, GolS2, and raffinose synthase 2 (BnRS2) in Arabidopsis and increased the enzymatic activity of GolS and RS and the concentration of raffinose family oligosaccharides (RFOs). Additionally, the overexpression lines exhibited increased antioxidant abilities. In contrast, the Arabidopsis mutant athsfa4a was more sensitive to dehydration, showing decreases in the efficiency of DT re-establishment, RFO contents, and oxidation resistance. Complementation analysis indicated that DT was rescued in athsfa4a/BnHSFA4a seeds to similar levels compared with those of Col-0. Taken together, these results indicated that BnHSFA4a probably functions in the regulation of GolS expression and activity, and activation of the antioxidative system and other stress response factors to improve DT.

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ABSTRACT. Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the 16841 Transformation and root formation of Brassica napus ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 16840-16855 (2015) hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD 600 = 0.5) for 3 to 5 min, followed by 2 days of cocultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features.

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