A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus

RNA interference (RNAi) or gene silencing is a natural defence response of plants to invading viruses. Here, we applied this approach against pepino mosaic virus (PepMV) isolates in their natural host, tomato. PepMV isolates differ in their genetic sequences, the severity of the disease they induce, and their worldwide distribution. PepMV causes heavy crop losses, mainly due to impaired tomato fruit quality. Resistant varieties are not yet available, despite many years of resistance breeding efforts within the tomato seed industry. To generate broad resistance to PepMV strains, conserved sequences from three different strains of PepMV (US1, LP, and CH2) were synthesized as a single insert and cloned in a hairpin configuration into a binary vector, which was used to transform tomato plants. Transgenic tomato lines that expressed a high level of transgene-siRNA exhibited immunity to PepMV strains, including a new Israeli isolate. This immunity was maintained even after graft inoculation, in which a transgenic scion was grafted onto nontransgenic infected rootstocks. However, an immune transgenic rootstock was unable to induce resistance in a nontransformed scion. These results provide the first example of engineered immunity to diverse PepMV strains in transgenic tomato based on gene silencing.     

In vitro direct plant regeneration and Agrobacterium‐mediated transformation of lucerne (Medicago sativa L.)

In vitro direct plant regeneration of lucerne was achieved by simultaneous application of thidiazuron (TDZ) and 6‐benzyladenine (BA) in Murashige and Skoog (MS) medium. Seedlings were germinated and grown for 6 d on growth regulator–containing MS medium. The shoot tip, consisting of the apical meristem along with parts of the cotyledonary leaves and hypocotyl, was then cultured on a medium containing the growth regulator(s). Adventitious budding of the shoot tip was promoted synergistically by treatment with TDZ and BA, and a maximum of thirty‐five shoots per explant was obtained on a medium supplemented with 2 mg L^?1 TDZ and 1 mg L^?1 BA. Plant regeneration frequency varied from 67 to 93%, and five Indian lucerne cultivars responded well to the regeneration protocol. The Agrobacterium‐mediated transformation frequency from co‐cultivated explants was 13% following multiple shoot induction. Southern analysis of the T₀ plants and T₁ progenies confirmed stable inheritance of the hpt marker gene. Agrobacterium infection of the explant caused a significant reduction in the plant regeneration frequency (23%) and the number of shoots induced (11) when compared with uninfected explants. A single shoot tip provided sufficient material to regenerate and establish twenty‐seven lucerne plants, whereas only nine plants could be regenerated from an Agrobacterium co‐cultivated explant. This transformation protocol could represent a valuable improvement over existing ones for lucerne.     

Application of Native Lactic Acid Bacteria (LAB) for Fermentative Recovery of Lipids and Proteins from Fish Processing Wastes: Bioactivities of Fermentation Products

Fermentation using native lactic acid bacteria (LAB) was evaluated for its effectiveness in recovering lipids and proteins simultaneously from freshwater fish visceral waste (FVW). Five different LAB isolated from fish processing waste were employed in a fermentation process that involved 10% (w/w) glucose, 2% (w/w) NaCl, and 10% (v/w) LAB. Cultures evaluated included four native isolates (Pediococcus acidilactici NCIM5368, Enterococcus faecalis NCIM5367, Pediococcus acidilactici FM37, and Pediococcus acidilactici MW2) from FVW with E. faecium NCIM5335 as the reference culture. Fermentation with native LAB resulted in recovery of > 90% oil present in the material as against no recovery in case of raw (unfermented) viscera and resulted in > 50% of degree of hydrolysis of proteins. The fatty acid profile of lipids was not affected by the fermentation process. The fermentation liquor, rich in hydrolyzed protein, exhibited antioxidant as well as antagonistic properties against several bacterial pathogens. The results clearly demonstrate the usefulness of fermentation using native isolates for simultaneous recovery of lipids and proteins from fish processing waste. It also asserts the value of fermentation as an eco-friendly method and aids in minimizing disposal/pollution problems associated with these solid wastes.     

Laboratory Wet Milling of Corn: Milling Fraction Correlations and Variations Among Crop Years

Several coproducts result from fractionating corn in the wet‐milling process. Because small changes in product composition and milling characteristics can have a major impact on coproduct yields and values, testing is done to anticipate final product yields. Using small sample size and controlled conditions, a laboratory wet‐milling method proved to be a useful tool for wet milling and genetics industries. A wet‐milling process (100‐g batches) was used for data collection. Data collected during 11 years (1994–2004) were observed for samples used as benchmarks to verify process precision and accuracy and determine correlations among wet‐milling yields. More than 400 milling tests were performed on benchmark samples. Data from benchmark samples also were pooled. Coefficients of variation were low (<6%) for mean yields; year‐to‐year standard deviations of benchmark sample yield means were homogenous and implied precision of the procedure. Some differences were detected in mean yields among years (P ≤ 0.05) for benchmark data due to combined effects of hybrid and environment. A negative correlation (r = –0.58) was observed between starch and gluten yield for pooled benchmark data. Four years (2002–2005) of milling data from commercially available hybrids were analyzed using the milling procedure. For pooled commercial data, the correlation between starch and fiber yield was (r = –0.80); correlation between starch and gluten was (r = –0.76).     

λ-cyhalothrin and cypermethrin induced in vivo alterations in the activity of acetylcholinesterase in a freshwater fish, Channa punctatus (Bloch)

In the present study, the in vivo effects of λ-cyhalothrin and cypermethrin on the activity of acetylcholinesterase (AChE, EC 3.1.1.7) were evaluated for 96 h in brain, muscle and gills of Channa punctatus. Both compounds exhibited tissue specific as well as dose dependent decrease in the activity of AChE. The treated fish showed a significant decrease in the activity of AChE in brain and a lesser inhibition in muscle and gills in response to the increasing concentrations of λ-cyhalothrin as well as cypermethrin. Our results indicated that the brain was the main target organ for both insecticides, followed by muscle and gills, as determined by AChE inhibition study. However, these organs showed variations in the degree of AChE inhibition for separate treatments of both insecticides. The λ-cyhalothrin was a more potent AChE inhibitor as compared to cypermethrin. These findings indicated that apart from the established mechanism of delayed closure of sodium ion channels, these pyrethroids inhibit the activity of AChE in C. punctatus which could further aggravate their neurotoxic effects.     

Comparative evaluation of fermented and non-fermented de-oiled rice bran with or without exogenous enzymes supplementation in the diet of <Emphasis Type="Italic">Labeo rohita</Emphasis> (Hamilton, 1822)

A 60-day feeding trial was conducted to study the effect of exogenous enzymes (xylanase and phytase) supplementation in the non-fermented and fermented de-oiled rice bran (DORB)-based diet of Labeo rohita. Four test diets (T1-DORB-based diet, T2-fermented DORB-based diet, T3-phytase and xylanase supplemented DORB-based diet, and T4-phytase and xylanase supplemented fermented DORB-based diet) were formulated and fed to the respective groups. Test diets T3 and T4 were supplemented with 0.01% xylanase (16,000 U kg^?1) and 0.01% phytase (500 U kg^?1) enzymes. One hundred twenty juveniles of L. rohita, with an average weight 5.01?±?0.02 g, were stocked in 12 uniform size plastic rectangular tanks in triplicate with 10 fishes per tank following a completely randomized design (CRD). Exogenous enzyme supplementation to the T3 group significantly improved the growth performance of L. rohita (p?<?0.05). Fermented DORB fed groups registered significantly lower growth irrespective of the supplementation of exogenous enzymes. The carcass composition (except CP %), enzyme activities (except amylase activity), globulin, and A/G ratio did not vary significantly (p?>?0.05). Based on the results of the present study, it is concluded that exogenous enzyme supplementation significantly increases the growth of fish fed with DORB-based diet.     

Inhibition of Iron Induced Lipid Peroxidation and Antioxidant Activity of Indian Spices and Acacia In Vitro

The spices used in the Indian foods such as Star anise (Illicium verum), Bay leaves (Cinnamomum zeylanicum) and Cobra’s saffron (Mesua ferrea), and Acacia (Acacia catechu), which have medicinal value, were used as test samples, to find their effect on in vitro lipid peroxidation (LPO). Rat liver post mitochondrial supernatant (PMS) in Tris HCl buffer, pH 7.4 was incubated for 0 and 1 h, with various test extracts in three different oxidant systems. The results show that addition of test samples to FeCl₃ medium at 0 h significantly stop the initiation of the LPO. However, the propagation phase of LPO was inhibited by Cobra’s saffron and Acacia and not by Star anise and Bay leaves. The test samples also showed strong reducing power and superoxide radical scavenging activity. Cobra’s saffron and Acacia showed the highest antioxidant activity, probably due to the higher polyphenol content as compared to other test samples.     

Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete,highly accumulating siRNA species

Virus resistance can be effectively generated in transgenic plants by using the plant’s silencing machinery. To study the specificity of gene-silencing-based resistance, homozygous tobacco (Nicotiana tabacum L.) plants containing a 597-nt hairpin RNA construct of the Potato Virus Y (PVY) replicase sequence were challenged with a variety of PVY strains. The transgene-carrying tobacco line was immune to five potato PVY strains with high sequence similarity (88.3–99.5%) to the transgene. Infection with more distant tomato and pepper PVY field strains (86–86.8% sequence similarity) caused delayed symptom appearance in the transgenic tobacco. Transgene production of small interfering (si) RNA was detected by northern blot and measured using a custom-designed microarray for the detection of small RNAs. siRNA accumulation peaks were observed throughout the inverted-repeat transgene. In the resistance-breaking tomato and pepper strains there were nucleotide differences in the sequences correlated to siRNA transgene accumulation, indicating the role of siRNA specificity in resistance breaking. The log of transgene siRNA signal intensity increased with probe GC content, indicating that the accumulating siRNA molecules were GC-rich. Sequence similarity of highly accumulating siRNAs with the target virus strain appears to be important for both resistance and resistance-breaking characteristics.     

Influence of duration of aeration on growth and survival of carp fingerlings during high density seed rearing

Impact of varied durations of aeration on growth, survival and production performances of catla (Catla catla), rohu (Labeo rohita), fringe lipped carp (Labeo fimbriatus) and olive barb (Puntius sarana) were evaluated in outdoor concrete tanks (10 m × 5 m × 1.3 m) during fingerlings rearing. At a combined stocking density of 0.5 million fry/ha, provision of night time aeration for 4, 8 and 12 h was evaluated as the three treatments, T-I, T-II and T-III, respectively against control (T-c, without aeration). Overall survival and net biomass of the species increased among the treatments significantly as a function of aeration hours (T-c < T-I < T-II < T-III). Aeration for 8 and 12 h showed significantly higher survival, harvested body weight and SGR in all the carps than those with 4 h (T-I) and no aeration (T-c), suggesting 8–12 h of aeration requirement for fingerlings rearing at such high density. Further, no significant difference between survival levels in T-II and T-III groups suggested 8 h of aeration to be adequate. However, while species-wise performance showed 8–12 h night time aeration to be advantageous for catla and rohu, aeration for 4 h was adequate for fringe lipped carp and olive barb.     

A novel approach to denitrification processes in a zero-discharge recirculating system for small-scale urban aquaculture

This paper presents an innovative process to solve the nitrate build-up problem in recirculating aquaculture systems (RAS). The novel aspects of the process lie in a denitrification bioreactor system that uses solid cotton wool as the primary carbon source and a unique degassing chamber. In the latter, the water is physically stripped of dissolved gaseous O₂ (by means of a Venturi vacuum tube), and the subsequent denitrification becomes more efficient due to elimination of the problems of oxygen inhibition of denitrification and aerobic consumption of cotton wool. The cotton wool medium also serves as a physical barrier that traps organic particles, which, in turn, act as an additional carbon source for denitrification. Operation in the proposed system gives an extremely low C/N ratio of 0.82 g of cotton wool/g of nitrate N, which contributes to a significant reduction of biofilter volume. The additional advantage of using solid cotton wool as the carbon source is that it does not release organic residuals into the liquid to be recycled. Operation of the system over a long period consistently produced effluents with low nitrate levels (below 10 mg N/l), and there was only a very small need to replace system water. The overall treatment scheme, also incorporating an aerobic nitrification biofilter and a granular filtration device, produced water of excellent quality, i.e., with near-zero levels of nitrite and ammonia, a sufficiently high pH for aquaculture, and low turbidity. The proposed system thus provides a solution for sustainable small-scale, urban aquaculture operation with a very high recovery of water (over 99%) and minimal waste disposal.