Optimising parameters for biolistic gun-mediated genetic transformation of tea [Camellia sinensis (L.) O. Kuntze]

Summary

Genetic improvement of tea through breeding is difficult. Therefore, transgenic tea plants expressing the osmotin gene from Nicotiana tabacum were produced using parameters optimised for biolistic-gun mediated transformation. During optimisation, a total of 4,500 somatic embryos were bombarded using nine combinations of variable target distances and burst pressures, while keeping the gap distance (0.6 cm) and macrocarrier flight distance (16 mm) constant.A total of 90 independent, PCR-positive lines were generated. Southern hybridisation confirmed integration of the osmotin gene in 26 out of 27 PCR-positive lines (three independent lines from each of the nine parameter combinations were selected at random). Statistical analysis revealed that the efficiency of transgene integration was significantly affected by target distance. Only those lines derived from somatic embryos bombarded with 1.0 µg plasmid DNA using a 7.58 MPa burst pressure and 9-cm target distance showed osmotin expression. This was evident from strong northern hybridisation and RT-PCR signals. Leaves of 4-year-old transgenic plants growing in a contained polythene tunnel showed improved osmotic adjustment in response to osmotic stress imposed by NaCl. The osmotic potentials of transgenic leaves immersed in 100 mM or 200 mM NaCl solutions were more negative than those of non-transformed control leaves.     

Shrimp Oil Extracted from Shrimp Processing By-Product Is a Rich Source of Omega-3 Fatty Acids and Astaxanthin-Esters,and Reveals Potential Anti-Adipogenic Effects in 3T3-L1 Adipocytes

The province of Newfoundland and Labrador, Canada, generates tons of shrimp processing by-product every year. Shrimp contains omega (n)-3 polyunsaturated fatty acids (PUFA) and astaxanthin (Astx), a potent antioxidant that exists in either free or esterified form (Astx-E). In this study, shrimp oil (SO) was extracted from the shrimp processing by-product using the Soxhlet method (hexane:acetone 2:3). The extracted SO was rich in phospholipids, n-3 PUFA, and Astx-E. The 3T3-L1 preadipocytes were differentiated to mature adipocytes in the presence or absence of various treatments for 8 days. The effects of SO were then investigated on fat accumulation, and the mRNA expression of genes involved in adipogenesis and lipogenesis in 3T3-L1 cells. The effects of fish oil (FO), in combination with Astx-E, on fat accumulation, and the mRNA expression of genes involved in adipogenesis and lipogenesis were also investigated. The SO decreased fat accumulation, compared to untreated cells, which coincided with lower mRNA expression of adipogenic and lipogenic genes. However, FO and FO + Astx-E increased fat accumulation, along with increased mRNA expression of adipogenic and lipogenic genes, and glucose transporter type 4 (Glut-4), compared to untreated cells. These findings have demonstrated that the SO is a rich source of n-3 PUFA and Astx-E, and has the potential to elicit anti-adipogenic effects. Moreover, the SO and FO appear to regulate adipogenesis and lipogenesis via independent pathways in 3T3-L1 cells.     

Effects of dietary supplements of selenium, vitamin E or combinations of the two on antibody responses of broilers

1. The effects of selenium and vitamin E supplementation on some immune parameters were investigated in broilers. 2. Broiler chicks were fed on maize-soybean diets with different concentrations of vitamin E (0-200 mg/kg) and selenium (0-0.2 mg/kg diet) either alone or in combinations from 1 to 42 d of age. 3. Chicks were immunised against Newcastle disease virus (NDV) vaccine at 21 d of age and haemagglutination inhibition (HI) titres were determined after 10 d. 4. Chicks receiving supplements of 200 mg vitamin E/kg and 0.2 mg selenium/kg produced significantly higher HI antibody titres. This was associated with an increased serum concentration of total immunoglobulins and circulatory immune complexes. 5. The chicks given 200 mg vitamin E/kg and 0.2 mg selenium/kg had significantly heavier spleen and bursa. 6. These results suggested that vitamin E and selenium have synergistic effects on immune responses.     

Assessment of genetic diversity in cabbage cultivars using RAPD and SSR markers

Genetic relationship and diversity among seven cabbage cultivars were analyzed using RAPD and SSR markers. These cultivars are of great commercial value in India and are confirmed for their reaction to black rot caused by Xanthomonas campestris pv. campestris. However, so far the extent of genetic diversity and relatedness has not been studied in these cultivars. A total of 17 selected RAPD primers generated 90 bands, 76 of which were polymorphic (84.44%). In addition, 27 selected SSR primers generated 67 amplified bands with 59 of which were polymorphic (87.6%). Though both the marker techniques were able to discriminate the cultivars effectively, analysis of combined data of markers (RAPD and SSR) resulted in better distinction of cultivars. By combining both the markers, a total of 157 bands were detected of which 135 bands (85.98%) were polymorphic, i.e. an average of 5.95 bands per primer. High level of polymorphism (> 85%) recorded with two different marker systems indicated a high level of genetic variation existing among the cultivars. Genetic relationship estimated using similarity co-efficient (Jaccard’s) values between different pairs of cultivars varied from 0.21 to 0.77 in RAPD, 0.42 to 0.82 in SSR, and 0.43 to 0.89 with combined markers. A high correspondence had been recorded between the values of genetic variations generated by UPGMA, clustering, and scatter plot diagrams. The cultivars ‘January King Sel. Improved’ and ‘Golden Acre’ are highly divergent cultivars as demonstrated by both the marker systems.     

Characteristics of apparent derivatives of the 2512 strain of infectious bursal disease virus when used as vaccines

The 2512 infectious bursal disease virus (IBDV) strain maintained in the authors' laboratory was compared with apparent 2512 IBDV isolates designated I-2512, P-2512, and H-2512. The latter three viruses were obtained from different sources and, ostensibly, had their origin in the Purdue laboratory. Their effects on immunogenicity, transmissibility, pathogenicity, and cell cultures varied. One of these isolates was said to be only 2 embryo passages higher than the original seed virus in our laboratory. Included in the study, also for comparison, was a cell-cultured-adapted 2512-cloned attenuated virus. The findings emphasize changes that may occur in the identity of a virus from manipulation, mutation, storage, errors in labeling, or other factors. These characteristics should be identified if the virus is to be used as a vaccine, in research, or for other purposes. The need for well-characterized reference strains in repositories is discussed relative to their importance in potential vaccine research and development.     

Anti-Chikungunya Viral Activities of Aplysiatoxin-Related Compounds from the Marine Cyanobacterium Trichodesmium erythraeum

Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC₅₀ values of 1.3 μM and 2.7 μM, respectively, and selectivity indices of 10.9 and 9.2, respectively.     

Molecular data from up to 130-year-old herbarium specimens do not support the presence of cherry powdery mildew in Australia

A strain of Podosphaera clandestina has been highlighted as a priority pest threat to the Australian cherry industry. Australia currently has no records of powdery mildew on cherry (Prunus avium). P. clandestina is reported to cause disease on a range of Rosaceae genera including Crataegus and Prunus; in Australia, P. clandestina has only been recorded on Crataegus. A recent species revision identified Podosphaera cerasi on P. avium as a separate species from P. clandestina. Therefore, a revision of which powdery mildew species is present in Australia on Crataegus is required to inform Australian plant biosecurity. Reference collection specimens from the Victorian Plant Pathology Herbarium (VPRI) recorded as Podosphaera spp. collected between 1889 to 2008 on cherry and three other host plant genera from Australia and overseas were sampled for DNA extraction and next-generation sequencing (NGS). Sequence data from preserved specimens were successfully mapped to internal transcribed spacer (ITS) sequences of P. clandestina in the strict sense, P. cerasi, and Podosphaera prunicola, and chloroplast matK sequences were used to identify plant hosts. Australian specimens on Crataegus hosts were P. clandestina in the strict sense and specimens on Prunus from the USA were identified as P. cerasi and P. prunicola. The outcome of this study confirmed the powdery mildew on Australian Crataegus specimens to be P. clandestina and none of the cherry powdery mildews (Podosphaera pruni-avium, P. cerasi, or P. prunicola) are present on Australian specimens in the VPRI collection, which suggests they are not present in Australia.