Enhancing somatic embryogenesis in avocado (Persea americana Mill.) using a two-step culture system and including glutamine in the culture medium

The development of a more efficient in vitro regeneration system for somatic embryos (SEs) of avocado (Persea americana) would facilitate the development of new superior cultivars for this valuable horticultural crop. In this study, we report a new and efficient method for maintenance and regeneration of avocado SEs. Avocado SEs of four cultivars remained healthy and viable in vitro for 11 months on a medium used for mango somatic embryogenesis, compared with 3-4 months on Murashige and Skoog medium. Various supplements and media modifications were investigated to improve the low conversion rate of regenerated plants from avocado SEs reported previously. The one-step system for regeneration of white-opaque somatic embryos (WOSEs) used solid medium only over a period of 12-14 weeks (sub-culturing every 6 weeks). Addition of praline and glutamine improved the total regeneration from 0 to 17.5% and 10.5%, and plant/shoot recovery from 0 to 12.5% and 5%, respectively. A two-step culture system involving the transfer of WOSEs of cultivar 'Reed' after 6 weeks on solid to liquid medium for 12-15 days as an intermediate step, followed by subculturing again onto solid medium for 6 weeks improved total regeneration to 29% and plant/shoot recovery to 18.3 from 0% when regenerated by subculturing on solid medium only. Supplementation with proline in the solid as well as liquid medium in the two-step culture system at 0.4 g/L increased total regeneration to 35% and plant/shoot recovery to 20%. We were able to achieve highest regeneration using glutamine at 1 g/L in the two-step culture system in terms of both total regeneration (58.3%, including 43.3% bipolar regeneration) and plant/shoot recovery (36.7%) rates, which were significantly higher than in any other treatment investigated. (C) 2013 Elsevier B.V. All rights reserved.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.

Sperm protamine deficiency correlates with sperm DNA damage in Bos indicus bulls

The primary purpose of spermatozoa is to deliver the paternal DNA to the oocyte at fertilization. During the complex events of fertilization, if the spermatozoon penetrating the oocyte contains compromised or damaged sperm chromatin, the subsequent progression of embryogenesis and foetal development may be affected. Variation in sperm DNA damage and protamine content in ejaculated spermatozoa was reported in the cattle, with potential consequences to bull fertility. Protamines are sperm-specific nuclear proteins that are essential to packaging of the condensed paternal genome in spermatozoa. Sperm DNA damage is thought to be repaired during the process of protamination. This study investigates the potential correlation between sperm protamine content, sperm DNA damage and the subsequent relationships between sperm chromatin and commonly measured reproductive phenotypes. Bos indicus sperm samples (n = 133) were assessed by two flow cytometric methods: the sperm chromatin structure assay (SCSA) and an optimized sperm protamine deficiency assay (SPDA). To verify the SPDA assay for bovine sperm protamine content, samples collected from testis, caput and cauda epididymidis were analyzed. As expected, mature spermatozoa in the cauda epididymidis had higher protamine content when compared with sperm samples from testis and caput epididymidis (p < 0.01). The DNA fragmentation index (DFI), determined by SCSA, was positively correlated (r = 0.33 ± 0.08, p < 0.05) with the percentage of spermatozoa that showed low protamine content using SPDA. Also, DFI was negatively correlated (r = -0.21 ± 0.09, p < 0.05) with the percentage of spermatozoa with high protamine content. Larger scrotal circumference contributes to higher sperm protamine content and lower content of sperm DNA damage (p < 0.05). In conclusion, sperm protamine content and sperm DNA damage are closely associated. Protamine deficiency is likely to be one of the contributing factors to DNA instability and damage, which can affect bull fertility. © 2014 American Society of Andrology and European Academy of Andrology.