Comparative performance of Gamma Amino Butyric Acid (GABA) and 5-Hydroxytryptamine (5-HT) in the diet of larvae and post larvae of giant freshwater prawn, Macrobrachium rosenbergii: Effect of dose and route of administration on growth and survival

Three experiments were conducted to evaluate the effect of different dose and route of administration of 5-Hydroxytryptamine (5-HT) and Gamma Amino Butyric Acid (GABA) on growth, survival and pigmentation of giant freshwater prawn, Macrobrachium rosenbergii, larvae (experiment 1) and post larvae (experiment 2 and 3). In experiment 1 larva were stocked at 100 /L in seven different treatments with each of three replicates. The treatments were T1 (control), T2 (100 μg/ml of 5-HT bath exposure for 2 days), T3 (1 μM of GABA bath exposure for 2 days), T4 (0.5% 5-HT in feed), T5 (0.25% 5-HT in feed), T6 (0.5% GABA in feed) and T7 (0.25% GABA in feed). Highest growth, transformation rate, pigmentation and survival of larvae were recorded in T2 group in experiment 1. In experiment 2, post larvae were stocked at 70 per tank with 200 L of water for 45 days. This experiment consisted of 5 treatment groups with each of 3 replication viz. T1 (control), T2 (0.5% 5-HT in feed), T3 (0.25% 5-HT in feed), T4 (0.5% GABA in feed) and T5 (0.25% GABA in feed). At the end of this experiment growth was found to be higher in the control than the other treatment group showing inhibitory effect of 5-HT and GABA on growth of post larvae. To confirm the result, a third experiment was conducted for 30 days. Thirty numbers of post larvae having similar size were segregated from the previous experimental tanks (experiment 2) and stocked in the tanks of 100 L of water for experiment 3. All treatments were fed with control diet. There was similar growth pattern in all the treatments, which were higher (P < 0.05) than control, confirms the inhibitory effect of neurotransmitter in the diet of PL. Therefore, the overall results of the present study suggest that the bath treatment of neurotransmitter is superior to the dietary addition with regard to the growth, survival and pigmentation of M. rosenbergii larvae. 5-HT is more effective than the GABA for larvae of M. rosenbergii.     

Sowing soil water content effects on chickpea (Cicer arietinum L.): Seedling emergence and early growth interaction with genotype and seed size

Soil water content at sowing is an important determinant of chickpea seed emergence and early growth. The seed emergence and seedling growth of 9 kabuli and 11 desi chickpea genotypes sown and grown under four soil moisture contents (25, 50, 75 and 100% of field capacity) was measured in a glasshouse study. Across soil moisture treatments the day of first emergence was negatively correlated to all plant growth parameters, e.g. above-ground biomass and plant height (r = −0.83 to −0.94). At 50 and 25% field capacity the onset of seed emergence was delayed, and both the number of seeds that emerged and early growth were reduced. Within the 25% soil moisture treatment, genotypes with an early emergence had a high rate of emergence (as estimated by regression against time) and produced seedlings with a high specific leaf area. Genotypic differences were observed in the number of seeds that emerged, day of first emergence, and early growth across the soil moisture treatments. Differences in final emergence were not related to chickpea type (i.e. desi or kabuli) or seed size, but the kabuli types emerged later than desi types (P < 0.001). The response of early dry matter production and leaf area to soil moisture was quadratically proportional to seed size, while the plant height response was linearly proportional to seed size. As a consequence kabuli chickpeas generated a higher degree of early vigour than desi types as soil moisture increased until seed sizes become extremely large, whereas under low soil moisture, kabuli chickpeas will have a much smaller or possibly no early vigour advantage over desi types.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Gibberellin-deficient dwarfs in potato vary in exogenous GA3 response when thega 1 allele is in different genetic backgrounds

Gibberellins (GAs) are involved in internode elongation and other important processes such as seed germination, flowering, maturation, tuberization, and tuber dormancy. The discovery of GA-deficient mutants enabled further study of the role of these hormones in many plant processes. GA-deficient mutants lack the ability to produce adequate amounts of gibberellin for normal growth, resulting in a rosette type growth and short internodes. Thega ₁ mutant allele was introduced into various genetic backgrounds including differentSolanum species and ploidies. Diploid GA-deficient genotypes were obtained by crossing haploidSolanum tuberosum ssp.andigena withSolanum chacoense. The progeny was then bulked and intermated to produce F₂ individuals. Tetraploid GA-deficient genotypes were obtained by crossingS. tuberosum ssp.andigena withSolanum sucrense and withSolanum gourlayi. The two resulting progenies were then bulked and intermated. Diploid and tetraploid GA-deficient genotypes were grown on MS media containing different levels of gibberellin (GA₃). Plant height and visual observations were made as a way to assess the response of these genotypes to GA₃. Concentration of 0.1 µM GA₃ and lower failed to restore normal plant height in both diploid and tetraploid genotypes. Normal plant height was restored in most of the GA-deficient genotypes when concentrations between 0.8 and 1.2 µM GA₃ were used. We found some important differences between these genotypes: (1) the level of GA₃ to restore normal plant height varies among the GA-deficient genotypes, some needed more GA₃ than others to grow normally; (2) the time to respond to the presence of GA₃ in the media differs between the GA-deficient genotypes, (3) tetraploid genotypes exhibited normal growth and internode length in response to GA₃, while diploid genotypes tended to show a rosette-type growth at the apical end. These results suggest thatga ₁ mutants can be affected by a series of modifier genes and/or iso-alleles. The importance of variable response to GA among dwarf individuals is two fold: (1) experiments measuring GA response should choose and clonally multiply one genotype to ensure uniform optimal response to GA application; and (2) variation betweenga ₁ mutant phenotypes could be used to characterize GA-response modifier genes.     

Potato freezing injury and survival,and their relationships to other stress

Breeding and selection for frost resistant potatoes with high yield and good quality is very promising. This has been demonstrated in a frost resistance breeding program conducted at the International Potato Center, Lima, Peru. Yield trials were conducted using selected frost resistant clones at various Andean locations at elevations from 3,200 to 3,900 meters (14). During the growth season, several hail storms and frosts occurred at these locations. These freezes (?3°C to ?4°C) were very useful in elimination of the undesirable clones under natural conditions. Clones with yield from 1 to 2 kg/plant have been selected. A yield of 1 to 2 kg/plant was superior to most common cultivars grown in the Andes. The difference in frost resistance between a resistant and sensitive type of potato is about 3 to 4°C.Solanum tuberosum is a sensitive type which, in most cases, can survive at about ?2.0°C. Evidence indicates that the difference in resistance to freezing injury is due to the genotype’s capability to tolerate freeze-induced dehydration and to withstand the aggregation of proteins under freezing stress. It is likely that freezing injury is caused primarily by the loss of membrane integrity and, therefore, that membrane thermal stability is paramount in the tolerance to freezing stress.     

Application of calcium and nitrogen for mitigating heat stress effects on potatoes

This study was designed to investigate the effect of calcium and nitrogen application during heat stress on leaf calcium concentration, transpiration rate, membrane thermostability, and biomass accumulation and partitioning. Micropropagated Russet Burbank potato (Solanum tuberosum L). plants were transplanted into 20 L pots containing 1:1 (v/v) soil: perlite and exposed to 30/20C (D/N) temperatures for four weeks (weeks 9–12 after transplanting) in a controlled-environment growth room. The maximum temperature was maintained for 6 hr during the middle of the 14 hr photoperiod. The nutrition treatments were N before stress (NBS), N during stress (NDS) and Ca and N during stress (Ca+NDS). Calcium was supplied as Ca(NO₃)₂. All treatments received the same total amount of nitrogen. Native soil Ca level without amendment (550 mg Ca/kg soil) was sufficient for potato plant growth under normal temperatures. Plants given Ca and N during heat stress had the highest leaf Ca concentration and transpiration rate during and 2 weeks after conclusion of the heat stress period. When measured after 4 weeks of heat stress, area and fresh and dry weight of the most recently mature leaf was significantly greater in NDS and Ca+NDS plants compared to NBS plants. Cellular membrane thermostability (measured as ion leakage from heat-treated leaf disks) was not affected by any treatment prior to heat stress. However, leaf tissue from Ca+NDS plants exhibited significantly higher membrane thermostability compared to NBS plants after 2 and 4 weeks of heat stress. At harvest, NDS and Ca+NDS plants had significantly higher leaf/stem (fresh weight ratio) values compared to NBS plants. Also, Ca+NDS plants had significantly greater total tuber and biomass values than NBS and NDS plants. Results of this study suggest that some detrimental effects of heat stress on plant growth and stomatal function may be alleviated by Ca and N application during heat stress. The data also suggest that mitigation of heat stress by Ca and N application during heat stress may maintain plant productivity when optimum growing temperatures are restored.     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.