Molecular phylogeny in Indian Citrus L. (Rutaceae) inferred through PCR-RFLP and trnL-trnF sequence data of chloroplast DNA

The taxonomy and phylogeny of Indian Citrus is revisited using PCR-RFLP of the trnD-trnT and rbcL-ORF 106 regions as well as sequence data analysis of the trnL-trnF intergenic spacer region of cpDNA. The study was based on 50 accessions of Citrus genotypes, collected from wild, semi-wild and domesticated stocks. Of the 13 restriction enzymes (RE) used for restriction digestion of the polymerase chain reaction (PCR) amplicons, four (Hinf I, Msp I, Alu I, Hae III) generated 47 restriction fragments, of which 24 (51%) were polymorphic. PCR-RFLP data showed a genetic distance ranging from 0 to 0.79 among 50 accessions of Citrus, and a cluster analysis, based on Neighbor-Joining (NJ) method, placed all the accessions in eight major clusters. Analysis of trnL-trnF sequences from 23 representative accessions of Citrus showed a pair-wise sequence divergence rate in the range of 0–0.064. NJ, minimum evolution (ME) and maximum parsimony (MP) analyses of trnL-trnF sequences produced phylogenetic trees, which placed all the 23 accessions in five clusters. PCR-RFLP analysis resulted in a well resolved phylogenetic tree with branches supported by moderate to high bootstrap values, while the trnL-trnF sequence-based trees showed only moderate to low bootstrap support for the internal tree branches, indicating uncertain origin of some Citrus genotypes. This study shows that the trnL-trnF spacer sequence data can detect genetic variation in Indian Citrus genotypes, but the utility of the data in inferring phylogeny at intra and inter-specific levels is limited probably by factors such as hybridization, bud mutations, apomixis and polyploidy. However, PCR-RFLP and trnL-trnF data supported the recognition of C. maxima, C. medica, and C. reticulata as the basal species of edible Citrus.     

Effect of a cytochrome P450 inhibitor on abscisic acid biosynthesis and stomatal regulation in citrus trees in water-stressed conditions

The effect of diniconazole, an inhibitor of cytochrome P450, on drought tolerance was examined in ‘Shiranui’ [(Citrus unshiu Marc. × Citrus unshiu Osbeck) × Citrus reticulate Blanco] trees. Diniconazole treatment increased abscisic acid (ABA) concentration in leaves compared with untreated controls in water-stressed conditions after 20 days of water-stress treatment. Diniconazole significantly decreased the stomatal aperture at 9 h after application compared with untreated controls in water-stressed conditions. The photosynthetic rate decreased in water-stressed conditions; however, regardless of the earlier stomatal closure induced by diniconazole application, the decrease of photosynthetic rate was delayed by the application.     

Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices (Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.     

Characteristics of phosphorus uptake kinetics of poinsettia and marigold

In a previous experiment it has been found that maximum uptake rate (I_max), Michaelis constant (K_m), and minimum nutrient concentration (C_min) as plant physiological characteristics may be important for phosphorus (P) uptake in peat-substrate. Thus, variation of P uptake parameters was evaluated with a series of depletion studies for poinsettia (Euphorbia pulcherrima) and marigold (Tagetes patula) under fluctuating climatic conditions and different developmental stages.     

Floral induction (FI) in longan (Dimocarpus longan, Lour.) trees. III: Effect of shading the trees on potassium chlorate induced FI and resulting hormonal changes in leaves and shoots

Previous experiments demonstrated that treatment of longan trees with potassium chlorate (KClO₃) induces “off season floral induction” (FI) even in the absence of the naturally required cool temperature [Manochai, P., Sruamsiri, P., Wiriya-alongkorn, W., Naphrom, D., Hegele, M., Bangerth, F., 2005. Year around off season flower induction in longan (Dimocarpus longan, Lour.) trees by KClO₃ applications: potentials and problems. Sci. Hortic. 104, 379–390]. Potassium chlorate, however, cannot replace the presence of functional mature leaves and sufficient light intensity. Here we examined in more detail the effect of shade (about 10% of natural sunlight) on KClO₃ affected hormone concentrations/transport of leaves and shoot apical buds (SAB) and their interactions with FI.     

Effects of dietary caffeine on growth,body composition,somatic indexes,and cerebral distribution of acetyl‐cholinesterase and nitric oxide synthase in gilthead sea bream (Sparus aurata), reared in winter temperature

This study aims at examining the effect of caffeine administration on growth, feed efficiency, and consumption of sea bream (Sparus aurata), reared in winter temperatures. Moreover, it is questioned whether caffeine has a central action in the brain and its effects are partly mediated via central brain mechanisms. For this, we studied the influences of caffeine treatment on the cerebral pattern of the cholinergic neurotransmission and the novel neuromodulator nitric oxide (NO), by means of acetyl‐cholinesterase (AchE) and nitric oxide synthase (NOS) histochemistry. Five different diets containing 0.0, 0.1, 1.0, 2.0 and 5.0 g caffeine kg^?1 of diet were administrated to five groups of fish. Caffeine adversely affected sea‐bream growth at a concentration higher than 1 g kg^?1 diet and increased feed conversion ratio in the treatments of 2 and 5 g kg^?1 (P < 0.05). The daily consumption of feeds was similar to all groups, indicating that caffeine did not influence diet palatability. AChE‐ and NADPH‐diaphorase histochemistry showed densely labeled cells and fibers mainly in dorsal telencephalon, preoptic, pretectal, hypothalamic areas, optic tectum, reticular formation, cerebellum and motor nuclei. When compared with matched caffeine‐treated animals, no differences in the histochemical pattern and cell densities of cerebral AChE and NADPH‐diaphorase were found.     

Supplemental iron and phosphorus increase dietary inclusion of cottonseed and soybean meal in olive flounder (Paralichthys olivaceus)

A long‐term feeding experiment was conducted to investigate the use of cottonseed and soybean meal (CS) with iron and phosphorus supplements in diets for olive flounder (Paralichthys olivaceus). Olive flounder with an initial average size of 28.5 ± 0.35 g (mean ± SD) were divided into 15 groups (three tanks per dietary treatment) and fed 480 g kg^?1 crude protein diets in which each of five isonitrogenous diets was formulated to contain different levels of cottonseed/soybean meal (1 : 1) to replace fish meal (FM) with iron and phosphorus supplementations. The five experimental diets were as follows: diet 1 (control), 0%CS; diet 2, 20%CS; diet 3, 30%CS; diet 4, 30%CS + Fe&P; and diet 5, 40%CS + Fe&P. After 26 weeks of feeding trial, no significant differences were observed in weight gain, feed utilization and survival among all the treatments. The total gossypol accumulation in liver of fish fed diets supplemented with iron was significantly lower than that of fish fed diets without supplementation of iron. The results indicate that the addition of iron in diets could prevent the absorption of free gossypol. The findings in this study suggest that dietary supplements of iron and phosphorus could increase the inclusion of cottonseed and soybean meal for FM replacement in diets for marine fish species.     

Growth and body composition of juvenile white shrimp,Litopenaeus vannamei,fed different ratios of dietary protein to energy

A 10‐week feeding experiment was conducted to evaluate the effect of different protein to energy ratios on growth and body composition of juvenile Litopenaeus vannamei (initial average weight of 0.09 ± 0.002 g, mean ± SE). Twelve practical test diets were formulated to contain four protein levels (300, 340, 380 and 420 g kg^?1) and three lipid levels (50, 75 and 100 g kg^?1). Each diet was randomly fed to triplicate groups of 30 shrimps per tank (260 L). The water temperature was 28.5 ± 2 °C and the salinity was 28 ± 1 g L^?1 during the experimental period. The results showed that the growth was significantly (P < 0.05) affected by dietary treatments. Shrimps fed the diets containing 300 g kg^?1 protein showed the poorest growth. However, shrimp fed the 75 g kg^?1 lipid diets had only slightly higher growth than that fed 50 g kg^?1 lipid diets at the same dietary protein level, and even a little decline in growth with the further increase of dietary lipid to 100 g kg^?1. Shrimp fed the diet with 420 g kg^?1protein and 75 g kg^?1 lipid had the highest specific growth rate. However, shrimp fed the diet with 340 g kg^?1 protein and 75 g kg^?1 lipid showed comparable growth, and had the highest protein efficiency ratio, energy retention and feed efficiency ratio among dietary treatments. Triglycerides and total cholesterol in the serum of shrimp increased with increasing dietary lipid level at the same dietary protein level. Body lipid and energy increased with increasing dietary lipid level irrespective of dietary protein. Results of the present study showed that the diet containing 340 g kg^?1 protein and 75 g kg^?1 lipid with digestible protein/digestible energy of 21.1 mg kJ^?1 is optimum for L. vannamei, and the increase of dietary lipid level has not efficient protein‐sparing effect.     

Optimal dietary lipid level for large yellow croaker (Pseudosciaena crocea) larvae

A 30‐day feeding experiment was conducted in blue tanks (70 × 50 × 60 cm, water volume 180 L) to determine the effects of dietary lipid levels on the survival, growth and body composition of large yellow croaker (Pseudosciaena crocea) larvae (12 days after hatchery, with initial average weight 1.93 ± 0.11 mg). Five practical microdiets, containing 83 g kg^?1 (Diet 1), 126 g kg^?1 (Diet 2), 164 g kg^?1 (Diet 3), 204 g kg^?1 (Diet 4) and 248 g kg^?1 lipid (Diet 5), were formulated. Live feeds (Artemia sinicia nauplii and live copepods) were used as the control diet (Diet 6). Each diet was randomly assigned to triplicate groups of tanks, and each tank was stocked with 3500 larvae. During the experiment, water temperature was maintained at 23(±1) °C, pH 8.0 (±0.2) and salinity 25 (±2) g L^?1. The results showed that dietary lipid significantly influenced the survival and growth of large yellow croaker larvae. Survival increased with the increase of dietary lipid from 83 to 164 g kg^?1, and then decreased. The survival of larvae fed the diet with 83 g kg^?1 lipid (16.1%) was significantly lower than that of larvae fed other diets. However, the survival in larvae fed the diet with 16.4 g kg^?1 lipid was the highest compared with other artificial microdiets. Specific growth rate (SGR) significantly increased with increasing dietary lipid level from 83 to 164 g kg^?1 (P < 0.05), and then decreased. The SGR in larvae fed the diet with 164 g kg^?1 lipid (10.0% per day) was comparable with 204 g kg^?1 lipid (9.6% per day), but were significantly higher than other microdiets (P < 0.05). On the basis of survival and SGR, the optimum dietary lipid level was estimated to be 172 and 177 g kg^?1 of diet using second‐order polynomial regression analysis respectively.     

Ovarian histology of stunted pond-reared Macrobrachium rosenbergii females

The present study describes the ovarian histology of stunted freshwater prawn Macrobrachium rosenbergii. The ovarian maturation of stunted animals was examined and compared with similar‐aged normal females. Ten animals of the stunted group and each maturation stage of the normal group were sampled from the same pond and had their ovaries removed for histological analysis. Body weight, body length, ovarian weight and gonadosomatic index (GSI) were recorded for each female. The diameters of the different oocyte types were compared among groups through histological assessments. The ovarian histology of stunted M. rosenbergii females indicated that although the somatic growth is severely affected (7.6 g), some energy has been placed on the vitellogensis. Stunted females showed the simultaneous occurrence of previtellogenic, vitellogenic and mature oocytes in their ovarian tissue, but overall oocyte diameter and GSI (1.02%) were significantly affected when compared with normal females.