Germplasm appraisal of western Himalayan tea: a breeding strategy for yield and quality improvement

Western Himalayan tea represents the status of Geographical Indication in the form of “Kangra Tea” which is unique to the tea grown world over due to its delicate flavor and high quality. The tea germplasm resources of western Himalayan region include selections from the commercial tea estates, abandoned tea gardens of Kangra valley and elite tea clones of CSIR-IHBT, Palampur. Data were recorded for different morphological traits contributing to yield and the biochemical parameters were evaluated using standard HPLC procedures. Multivariate clustering of the data differentiated the germplasm resources into distinct groups based on comparisons among the clusters for leaf size and biochemical parameters. Leaf size differentiated the tea accessions into six phenotypic groups, while shoot density which is an important yield parameter was observed to be independent of leaf size. On the basis of total catechin and caffeine contents, tea accessions were differentiated into nine groups of which Group I in comparison with other groups, recorded the highest total catechin content, moderate to high caffeine level and high astringency factor (AF). Epicatechin gallate (ECG) had a significantly high correlation with AF, implying that high levels of ECG are critical along with corresponding levels of Epigallocatechin gallate (EGCG) for the production of Theaflavin 3,3′digallate (TFDG) which is an important quality constituent providing astringency and briskness to black tea liquor. Sustainability of tea breeding programme can be achieved through organization of germplasm resources and by utilizing elite tea genotypes in breeding programme.     

Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle,buffaloes, and goats

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.     

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.     

Crop performance in permanent raised bed rice–wheat cropping system in Punjab,India

Raised beds are widely used in agriculture in developed countries and have proven to be an excellent option for wheat. Permanent raised beds may also offer benefits for rice–wheat (RW) systems in South Asia, in terms of both production and the possibility that furrow-irrigation may be more efficient than flood irrigation. The performance of a RW system on permanent raised beds (37 cm wide, 15 cm high, furrow width 30 cm) was compared with conventional cultivation on the flat on sandy loam and loam soils in replicated experiments in central Punjab, India. The experiments commenced with wheat sown in November 2002, and were continued for 8 crops.     

Isolation, culturing and characterization of feeder-independent amniotic fluid stem cells in buffalo (Bubalus bubalis)

Heterogeneous amniotic fluid contains various cell types. The aim of this study was to characterize and differentiate some of the key stemness attributes of the amniotic fluid-derived cells in buffalo (Bubalus bubalis). The amniotic fluid (AF) cells were cultured without feeder cells, in DMEM containing 15% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin/ampicillin, 1% vitamin solution, and 1% l-glutamine in 5% CO(2) in humidified air at 38.5±0.5 °C. After 6 days of culture different types of cells viz., star shaped (62.7%), spherical without nucleus (1.9%), spherical with nucleus (26.4%), pentagonal (0.4%), and free floating/rounded cells (8.3%) were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. Expression of alkaline phosphatase (AP) and Oct-4, Nestin and FGF-5 were observed from the AF cells at different passages. Using species-specific primers, a PCR amplicon of 200, 296 and 210 bp were observed for Oct-4, Nestin and FGF-5, respectively. The cells were found to have a normal karyotype at different passages. These results may contribute towards establishing non-embryonic pluripotent stem cells for various therapeutic and reproductive biotechnological applications in the species.     

An early gene of the flavonoid pathway, flavanone 3-hydroxylase, exhibits a positive relationship with the concentration of catechins in tea (Camellia sinensis)

Tea (Camellia sinensis (L.) O. Kuntze) leaves are a major source of flavonoids that mainly belong to the flavan 3-ols or catechins. Apart from being responsible for tea quality, these compounds have medicinal properties. Flavanone 3-hydroxylase (F3H) is an abundant enzyme in tea leaves that catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. We report a full-length cDNA sequence of F3H from tea (CsF3H Accession no. AY641730). CsF3H comprised 1365 bp with an open reading frame of 1107 nt (from 43 to 1149) encoding a polypeptide of 368 amino acids. Expression of CsF3H in an expression vector in Escherichia coli yielded a functional protein with a specific activity of 32 nmol min(-1) mg protein(-1). There was a positive correlation between the concentration of catechins and CsF3H expression in leaves of different developmental stages. CsF3H expression was down-regulated in response to drought, abscisic acid and gibberellic acid treatment, but up-regulated in response to wounding. The concentration of catechins paralleled the expression data. Exposure of tea shoots to 50-100 microM catechins led to down-regulation of CsF3H expression suggesting substrate mediated feedback regulation of the gene. The strong correlation between the concentration of catechins and CsF3H expression indicates a critical role of F3H in catechin biosynthesis.     

Evaluation of a Suitable Extractant and Determination of Critical Limit of Boron in Soil and Mustard (Brassica Juncea L.) Plant by Various Extractants in Inceptisols of Varanasi

Pot culture experiment was conducted to evaluate the suitability of extractants and to determine the critical limit of boron (B) in soil and mustard plant in Inceptisols of Varanasi. Twenty-one bulk soil collected from different locations were used for growing mustard. Five extractants, namely hot water, hot 0.01molar (M) calcium chloride (CaCl₂), 0.01M CaCl₂ + 0.05 M mannitol, 1.0 M ammonium acetate (NH₄OAC) and 0.05 M hydrochloric acid (HCl), were assessed by correlating the amount of extractable B in untreated and B fertilizer-treated soil with Bray’s per cent yield, plant tissue B concentration and B uptake by mustard. Similarly, correlation coefficients of the B extracted by different extractants and soil properties were calculated. The suitability of B extracted by different extractants was in the order of hot 0.01M CaCl₂ (HCC-B) > hot water (HW-B) > 1.0 M NH₄OAC (AA-B) > 0.05M HCl (HA-B) > 0.01M CaCl₂ + 0.05M mannitol (CCM-B). The critical limits of extractable B in soil as determined by the graphical procedure were 0.54, 0.60, 0.36, 0.45 and 0.45 mg kg^?1 and the statistical procedures were 0.54, 0.60, 0.38, 0.46 and 0.48 mg kg^?1 with HW-B, HCC-B, CCM-B, AA-B and HA-B, respectively. Soil containing available B below the critical limit responded to B fertilization.     

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.