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.     

Antibacterial and Cytotoxic New Napyradiomycins from the Marine-Derived Streptomyces sp. SCSIO 10428

Three new napyradiomycins (1–3) were isolated from the culture broth of a marine-derived actinomycete strain SCSIO 10428, together with six known related analogues napyradiomycin A1 (4), 18-oxonapyradiomycin A1 (5), napyradiomycin B1 (6), napyradiomycin B3 (7), naphthomevalin (8), and napyradiomycin SR (9). The strain SCSIO 10428 was identified as a Streptomyces species by the sequence analysis of its 16S rRNA gene. The structures of new compounds 1–3, designated 4-dehydro-4a-dechloronapyradiomycin A1 (1), 3-dechloro-3-bromonapyradiomycin A1 (2), and 3-chloro-6,8-dihydroxy-8-α-lapachone (3), respectively, were elucidated by comparing their 1D and 2D NMR spectroscopic data with known congeners. None of the napyradiomycins 1–9 showed antioxidative activities. Napyradiomycins 1–8 displayed antibacterial activities against three Gram-positive bacteria Staphylococcus and Bacillus strains with MIC values ranging from 0.25 to 32 μg mL⁻¹, with the exception that compound 3 had a MIC value of above 128 μg mL⁻¹ against Staphylococcus aureus ATCC 29213. Napyradiomycins 2, 4, 6, and 7 exhibited moderate cytotoxicities against four human cancer cell lines SF-268, MCF-7, NCI-H460, and HepG-2 with IC₅₀ values below 20 μM, while the IC₅₀ values for other five napyradiomycins 1, 3, 5, 8 and 9 were above 20 μM.     

Screening of genotypes of menthol mint Mentha arvensis for high yields of herbage and essential oil under late cropping conditions of the sub-tropical Indo-Gangetic plains

Summary

The essential oil yields and expression of related characters were compared for seven cultivar genotypes of menthol mint Mentha arvensis using two methods of planting in the winter rabi – summer season (October to July) in a sub-tropical agroclimatic environment. The crops of all the cultivars were planted in the field by (1) sowing of suckers on 2 January and (2) transplanting germinated pieces of sucker at different times between 17 March to 14 April. Staggering of transplanting time up to 7 April did not affect oil yields and the related plant growth properties of mint crops. The oil yields of the crops planted on 14 April were lower by about 30%. In the early sucker planted crops, the oil yields were about 30% higher than those obtained from the transplanted crops of 17 March to 7 April and about double that obtained from crops transplanted on 14 April. The oil yields from the crops of the superior genotype Kosi were equal to or higher than the corresponding means of all genotypes under both planting methods. The oil yield from the crops of Kosi genotype obtained by sucker sowing method was estimated as 333 kg ha^–1. The corresponding average yield from the crops of this genotype obtained by transplanting of germinated suckers between 17 March and 7 April was about 293 kg ha^–1 and that from the crop transplanted on 14 April was 218 kg ha^–1. With the Kosi genotype, the latter two types of transplanted mint crops gave oil yields lower only by about 12% and 33% compared with the long-duration sucker-sown crop. It is concluded that a crop of mustard (brassica), Bengalgram (chickpea) or wheat sown between October and November and harvested between early March to middle of April could be taken before cropping of the Kosi genotype of M. arvensis by plantlet transplanting. These results demonstrate the potential of the following rotation of crops in the sub-tropical environments: from June/July/August to October/November (kharif cropping season), rice, maize, sorghum or pigeonpea; from November/December to February/March, mustard and Bengalgram or from November/December to April (rabi cropping season), wheat; from March/April to June/July (zaid cropping season), transplanted menthol mint.     

Comparison of seed-derived with micropropagated male-sterile plants of Tagetes erecta L. for F1 hybrid seed production

Summary

To multiply large number of male-sterile marigold plants for F₁ hybrid seed production, an efficient protocol for in vitro cloning of field-grown differentiated male sterile plants has been developed. A comparative field performance study of tissue culture and seed-derived male sterile plants of two marigold genotypes was undertaken to test the possibility of using micropropagated plants in hybrid seed production. Tissue culture raised plants of both genotypes had superior field performance to the seed-derived counterparts. These plants were more vigorous in growth, i.e. in terms of plant height, number of secondary branches and number of leaves and plant spread, while the leaf chlorophyll contents were equal to that of seedling plants. Flowering was earlier by 2-3 weeks and the number of flowers per plant was also higher in such plants. Repeated hand pollination of sterile flowers with bagged flowers of cv. Pusa Narangi Gainda showed that seed set and bold seed yield were higher or almost comparable with the seed-derived plants. The results clearly indicate that the tissue culture can be adopted for the successful cloning of male-sterile plants, which could then be utilized for producing F₁ seeds with higher quantities of bold seeds with better storability.     

Optimising parameters for biolistic gun-mediated genetic transformation of tea [Camellia sinensis (L.) O. Kuntze]

Summary

Genetic improvement of tea through breeding is difficult. Therefore, transgenic tea plants expressing the osmotin gene from Nicotiana tabacum were produced using parameters optimised for biolistic-gun mediated transformation. During optimisation, a total of 4,500 somatic embryos were bombarded using nine combinations of variable target distances and burst pressures, while keeping the gap distance (0.6 cm) and macrocarrier flight distance (16 mm) constant.A total of 90 independent, PCR-positive lines were generated. Southern hybridisation confirmed integration of the osmotin gene in 26 out of 27 PCR-positive lines (three independent lines from each of the nine parameter combinations were selected at random). Statistical analysis revealed that the efficiency of transgene integration was significantly affected by target distance. Only those lines derived from somatic embryos bombarded with 1.0 µg plasmid DNA using a 7.58 MPa burst pressure and 9-cm target distance showed osmotin expression. This was evident from strong northern hybridisation and RT-PCR signals. Leaves of 4-year-old transgenic plants growing in a contained polythene tunnel showed improved osmotic adjustment in response to osmotic stress imposed by NaCl. The osmotic potentials of transgenic leaves immersed in 100 mM or 200 mM NaCl solutions were more negative than those of non-transformed control leaves.     

Role of sucrose synthase and invertases during petal senescence in rose (Rosa hybrida L.)

Summary

The roles of sucrose synthase and invertases were explored in relation to petal senescence in rose (Rosa hybrida L.). A developmental shift in the activities of these enzymes was observed. Higher sucrose synthase activity (0.52 – 0.95 µmol sucrose min^–1 mg^–1 protein) was observed during the initial stages (S1 and S2) of flower bud development, in contrast to invertases. However, the lower activity (0.56 µmol sucrose min^–1 mg^–1 protein) of sucrose synthase at a later stage (S6) of senescence could help the mobilisation of vacuolar sucrose. The different isoforms of invertase exhibited variable levels of activity. Insoluble acid invertases (IAI) were the most active (11.01 µmol glucose min^–1 mg^–1 protein), followed by soluble acid invertases (SAI; 8.02 µmol glucose min^–1 mg^–1 protein), and soluble neutral invertases (SNI; 0.74 µmol glucose min^–1 mg^–1 protein) at Stage-4. A significant decline in invertase activities (IAI, 0.98; SAI, 1.25; SNI, 0.32 µmol glucose min^–1 mg^–1 protein) coincided with higher levels of ethylene production at the later stages (S5 and S6) of flower bud development and senescence. We propose that developmental as well as ethylene-mediated pathways account for petal senescence in rose.     

Isolation and characterization of gut bacteria of fruit fly, Bactrocera tau (Walker)

Gut bacteria of fruit fly, Bactrocera tau (Walker) (Diptera: Tephritidae), were isolated and the isolates attractive to B. tau adults were characterized using morphological, biochemical and 16S rRNA analyses to determine their taxonomic position. Based upon morphological, biochemical and 16S rRNA sequences (on the basis of closest match), five gut bacterial species of B. tau were characterized as Delftia acidovorans, Pseudomonas putida, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp., of which four bacterial isolates, viz., Delftia acidovorans, Flavobacterium sp., Defluvibacter sp. and Ochrobactrum sp. are new records from guts of the fruit fly species.