Hairpin-RNA mediated silencing of endogenous FAD2 gene combined with heterologous expression of Crambe abyssinica FAE gene causes an increase in the level of erucic acid in transgenic Brassica carinata seeds

The 3′-UTR of the FAD2 gene from Brassica carinata was cloned by PCR and used to prepare an intron-spliced hairpin RNA (ihpRNA) construct. Compared to that of the wild type (WT) background, this construct, when expressed in B. carinata, resulted in a high degree of FAD2 gene silencing accompanied by strong increases of up to 16 and 10% in oleic acid and erucic acid proportions, respectively. The increase in 18:1 was accompanied by a concomitant proportional reduction in 18:2. A second construct containing ihpRNA targeted to the endogenous FAD2 gene in addition to the heterologous Crambe abyssinica FAE gene under the control of seed specific napin promoter, was used to transform B. carinata. This approach resulted in an even greater increase in erucic acid proportions, by up to 16% in T₁ segregating seeds as compared to that of the WT control. To our knowledge, this is currently the highest accumulation of erucic acid achieved in B. carinata seeds using transgenic approaches, making it an increasingly-attractive alternative to high erucic B. napus cultivars as an industrial oil crop. Database: The nucleotide sequence reported in this paper has been submitted to the EMBL/GenBank under accession number DQ250814.     

Do seed mass and family affect germination and juvenile performance in Knautia arvensis? A study using failure-time methods

Germination and seedling establishment are vulnerable stages in the plant life cycle. We investigated how seed mass and family (progeny origin) affect germination and juvenile performance in the grassland herb Knautia arvensis. Seeds were produced by cross-pollination by hand. The fate of 15 individually weighed seeds from each of 15 plants was followed during a 3-month growth chamber experiment. Progeny origin affected germination, both through seed mass and as an independent factor. Two groups of progenies could be distinguished by having rapid or delayed germination. The two groups had similar mean seed masses, but a positive relationship between seed mass and germination rate could be established only among the rapidly germinating progenies. These biologically relevant patterns were revealed because timing of germination was taken into account in the analyses, not only frequencies. Time-to-event data were analysed with failure-time methods, which gave more stable estimates for the relation between germination and seed mass than the commonly applied logistic regression. Progeny origin and seed mass exerted less impact on later characters like juvenile survival, juvenile biomass, and rosette number. These characters were not affected by the timing of germination under the competition-free study conditions. The decrease in the effect of progeny origin from the seed and germination to the juvenile stages suggests that parental effects other than those contributing to the offspring genotype strongly influenced the offspring phenotype at the earliest life stages. Further, the division of progeny germination patterns into two fairly distinct groups indicates that there was a genetic basis for the variation in stratification requirements among parental plants. Field studies are needed to elucidate effects of different timing of germination in the seasonal grasslands that K. arvensis inhabits.     

Brood protection at a cost: mouth brooding under hypoxia in an African cichlid

This study quantifies the behavioral response of the widespread mouth brooding African cichlid Pseudocrenilabrus multicolor victoriae to progressive hypoxia. We exposed four gender/stage classes of P. multicolor (males, brooding females, females that had just released young, and non-brooding females) to progressive hypoxia and recorded the percent time spent using aquatic surface respiration (surface skimming, ASR) and gill ventilation rates. This was done for fish collected from three sites in Uganda (lake, swamp, and river) after long-term acclimation to normoxia. There was no effect of site of origin on response to hypoxia, but ASR thresholds did differ between gender/stage classes. The oxygen level (threshold) at which spent 10, 50, and 90% of their time at the surface using ASR was much higher for brooding females than for males, whereas ASR thresholds did not differ between non-brooding females and males. Similarly, the level at which ASR was initiated was much higher in brooding females than males, but did not differ between males and non-brooders, or between males and females than had just released young. The rate of gill ventilation dropped significantly in males and all stages of females after initiation of ASR, suggesting that surface skimming increases efficiency of oxygen acquisition. These results suggest that mouth brooding in female P. multicolor ASR improves oxygen uptake but imposes a cost in terms of time spent at the water surface, and this may affect maternal predation risk in low-oxygen habitats.     

Competition for territories does not explain allopaternal care in the tessellated darter

Extensive research has focused on understanding the evolution of parental care, with fishes providing important model systems for understanding patterns of variation within and between species. Classic theory predicts that individuals will care for offspring when the fitness benefits through increased offspring survival and growth outweigh the cost to the parents through decreased future reproductive opportunities. Yet, a puzzling observation not explained by this basic theory is the fact that in some species individuals defend and provision unrelated offspring and thus exhibit alloparental care. The tessellated darter, Etheostoma olmstedi, represents one of the first known examples of allopaternal care in fishes. In this species, males often clean and guard eggs fertilized but deserted by other males. Allopaternal care has been argued to occur in the tessellated darter because of competition for a limited number of mating sites where less dominant males accept territories with eggs when other breeding sites are not available. Here, we test this hypothesis using male territory choice experiments. When allowed to choose between two otherwise identical territories either containing eggs fertilized by another male or with no eggs, males spent significantly more time at territories with eggs. This demonstrates that competition for mating territories is not the primary factor explaining the existence of allopaternal care in the tessellated darter. Instead, males of this species may exhibit allopaternal care to dilute predator pressure on their own eggs or because females prefer to mate with males whose territories contain eggs.     

Hypocotyl-based <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.