Effect of Supplementation of Taurine or Trehalose in Extender on Immunolocalization of Tyrosine Phosphoproteins in Buffalo and Cattle (Karan Fries) Cryopreserved Spermatozoa

The present study assessed the effects of incorporation of Taurine or Trehalose in extender on immunolocalization of tyrosine phosphoproteins, Cryocapacitation and other sperm quality parameters (motility, viability and membrane integrity) in post‐thawed sperm from Buffalo (Murrah) and Cattle (Karan Fries). Six ejaculates from six individual bulls from both species were chosen at random and split into four aliquots: one aliquot without dilution (fresh sample), another diluted in egg yolk tris‐citrate (EYTC) extender and the rest of aliquots with EYTC dilution supplemented with taurine (50 mm ) or trehalose (100 mm ), respectively, and cryopreserved. Following cryopreservation, semen were thawed and assessed for standard semen quality parameters. Extent of capacitation in cryopreserved spermatozoa was measured by inducing in vitro acrosome reaction followed by dual staining. Immunolocalization of tyrosine phosphoproteins was carried out by immunocytochemistry using primary antibody clone pT‐154 (anti‐phosphotyrosine antibody) and FITC‐conjugated secondary antibody. Immunofluorescent signals were analysed for level of protein tyrosine phosphorylation in spermatozoa. Post‐thaw semen evaluation showed supplementation of taurine or trehalose to EYTC extender significantly (p < 0.05) increased motility, viability and membrane integrity of spermatozoa in both species. Percentage of cryocapacitated spermatozoa was significantly (p < 0.05) higher in cattle as compared to buffalo and degree of cryocapacitaion of spermatozoa decreased significantly (p < 0.05) upon supplementation of additives in both the species. It was also found that tyrosine phosphoproteins were localized differentially in fresh and cryopreserved spermatozoa. Supplementation of taurine or trehalose to freezing extender changed the localization of tyrosine phosphoproteins in cryopreserved spermatozoa similar to fresh in both the species. The results obtained clearly indicated that supplementation of taurine or trehalose to EYTC prior to cryopreservation improves Buffalo and Cattle sperm quality in terms of cryocapacitation and immunolocalization of tyrosine phosphoproteins during freezing–thawing process.     

Physiological Factors Limiting Grain Growth in Wheat Ears Cultured in Sucrose Solution

Detached wheat ears (15 days post-anthesis) were cultured in liquid nutrient medium containing different sucrose concentrations (2. 6 and 10%). The volume of solution taken up was highest from 2% sucrose solution and decreased with increasing sucrose concentration. Grain development in terms of grain weight, glumes and rachis weight, nitrogen and protein content showed a linear increase in these variables over 11 days. Dry weight per grain increased with the sucrose level in the medium and the reduced uptake of solution did not decrease the moisture percentage of the grain. The culture of ears decreased the solution pH more with 2% than with 10% sucrose. Addition of 0.5% chloramphenicol to the culture solution had no adverse effect on grain weight, rather it prevented contamination and maintained a higher pH of the solution.     

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.     

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non‐reproductive tissues

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high‐fertile (G1) and low‐fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real‐time PCR (Roche LC‐480). Our work shows that the relative quantification by RT‐PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT‐PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.     

Glycine max and Glycine soja are capable of cold acclimation

Soybean has been considered a cold intolerant species; based largely upon seed germination and soil emergent evaluations. This study reports a distinct acquisition of cold tolerance, in seedlings, following short acclimation periods. Diversity in cold responses was assessed in eight cultivars of Glycine max and six accessions of G. soja. All varieties of soybean significantly increased in freezing tolerance following acclimation. This study indicates soybean seedlings are indeed capable of sensing cold and acquiring cold tolerance. Germination rates after cold imbibition were negatively correlated with maturity group, but positively correlated with cold acclimation potential in G. soja. Seed fatty acid composition was varied between the species, with Glycine soja accessions containing about 2‐times more linolenic acid (18:3) than G. max. Furthermore, high levels of linoleic acid (18:2) in seeds were positively correlated with germination rates following cold imbibition in G. soja only. We suggest that domestication has not impacted the overall ability of soybean to cold acclimate at the seedling stage and that there is little variation within the domesticated species for ability to cold acclimate. Thus, this brief comparative study reduces the enthusiasm for the “wild” species as an additional source of genetic diversity for cold tolerance.     

Water use in rice crop through different methods of irrigation in a sodic soil

Sodic soils are characterized by high exchangeable sodium on exchange sites, soil pH greater than 8.5, relatively low electrical conductivity, low infiltration rate and dispersed clay. These characteristics restrict the capacity of soil to absorb water, resulting in poor infiltration. Evidently, these soils require application of irrigation water at shorter intervals for crop production. Thus, irrigation strategy for sodic soils differs from that of normal soils. An experiment to determine the suitable irrigation strategy along with methods of application namely: surface (farmer’s practice), sprinkler (double nozzle impact sprinkler), and low-energy water application device (LEWA) were initiated in the year 2012 for rice crop. Irrigation depths of 6 cm in case of surface method and 4 cm in case of sprinkler and LEWA methods were applied at each irrigation event. The irrigation events for rice were scheduled at 2-DAD (days after the disappearance of the ponded water), 3-DAD, and 4-DAD through surface method, and at daily, 1- and 2-day intervals (after initial ponding disappeared) by sprinkler and LEWA methods. Sprinkler and LEWA methods resulted in highest rice yield of 4.4 t ha^?1 in irrigated plots at the 2-day interval which was at par with the highest yielding surface-irrigated plot scheduled at 2-DAD. At the same time, irrigation strategy of 2-day interval through sprinkler and LEWA methods registered water saving to the extent of 30–40% over 2-DAD under surface irrigation method. Results revealed that there could be substantial saving of water and energy (electricity and diesel) through the use of sprinkling devices for irrigating rice under sodic soil environments.     

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R.?solanacearum has been classified into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.     

Survival and development of spotted bollworm, <Emphasis Type="Italic">Earias vittella</Emphasis> (Fabricius) (Lepidoptera: Nolidae) on different transgenic <Emphasis Type="Italic">Bt</Emphasis> and isogenic non-<Emphasis Type="Italic">Bt</Emphasis> cotton genotypes

Four Bt cotton hybrids, each with one of four different events, viz., MRC 6301 Bt (cry1Ac gene), JKCH 1947 Bt (modified cry1Ac gene), NCEH 6R Bt (fusion cry1Ac/cry1Ab gene) and MRC 7017 Bollgard II (cry1Ac and cry2Ab genes) were compared for survival and development of Earias vittella (Fabricius) along with their isogenic non-Bt genotypes. None of the neonates were able to complete the larval period and reach pupal stage on squares of 90, 120 and 150 days old crop of all Bt hybrids. Likewise, on bolls also, zero per cent larval survival was observed in all Bt hybrids except JKCH 1947 Bt where 0.67 per cent larvae could manage to reach pre-pupal stage at 120 and 150 days old crop but failed to form cocoon and enter pupal stage. The surviving larva took more development time (3.7 to 5.4 days) as compared to larvae fed on bolls of JKCH 1947 non-Bt. The average survival period (ASP) of larvae was in order of 150 > 120 > 90 days old crop among the crop ages; JKCH 1947 Bt > MRC 6301 Bt > NCEH 6 R Bt > MRC 7017 Bollgard II among Bt hybrids; and bolls > squares between fruiting bodies. However, reverse was true for speed index of toxic effect. The concentration of Cry toxin varied significantly in squares and bolls and also among the crop ages. The amount of Cry toxin in squares and bolls had significant negative correlation with ASP of the E. vittella larvae.     

Callus Initiation in the Propagation of Papaya (Carica Papaya L.) in Vitro

Papaya tissue excised from a sexually differentiated plant could not proliferate in vitro due to the exudation of latex. Stem segments of young papaya seedlings successfully formed callus on a modified Murashige and Skoog’s medium supplemented with NAA.