Development of simplified and rapid nucleic acid release protocol for PCR based detection of <Emphasis Type="Italic">Potato viruses</Emphasis>

A simple, cost-effective and rapid viral nucleic acid release (NAR) buffer suitable for RT-PCR based diagnostic assay was developed for the detection of potato viruses. The NAR buffer and commercially available RNA isolation kit were compared for RT-PCR based assay, where an amplicon of expected size (~380 bp) targeting PVY was observed in both isolations indicating that it can be used in RT-PCR based diagnostic assays. The same was further validated for its repeatability by running across more than hundred suspected potato leaf samples collected from different sources where, it showed consistent results for the presence of PVY indicating its reliability. The NAR buffer assay was examined for its sensitivity in comparison with the kit based isolation where both the assays were able to detect even up to 10^?5 dilution without affecting the sensitivity. NAR buffer was found stable up to 28 days at -20 °C and for 14 days at 4 °C without losing PCR sensitivity. The assay was also found effective to release the nucleic acid from potato leaves, thrips and aphids for PCR and RT-PCR based detection of DNA viruses like ToLCNDV-potato and other RNA viruses. The developed protocol is simple, less laborious, time-saving (10-15 min) and economical (1/100th of kit) as compared to kit based protocol. The assay can be adopted in diagnostic laboratories for detection of RNA/DNA viruses from potato plants and in thrips as well.     

Core subsets of base collections and priorities of national programmes: Indian perspective

Summary The establishment of representative core collections and back-up reserve collections was proposed to facilitate effective management and to promote utilization of large base collections. The priorities of the developing national PGR programmes, in the organization of components of their base collections, are different from those of IARCs since the strengths as well as limitations of the two systems vary. Unlike most IARCs, national programmes have the option of networking their active collections, maintained at several eco-sites, and linking this network to the base collection kept under long-term storage. This keeps open the option to develop situation-specific subsets for an effective germplasm utilization. There is a pressing need for a scientific re-examination of the concept of germplasm core in order better to apply it in developing core subsets in the national PGR programmes. The formulation of situation-specific subsets is advocated, as the system would be directed to users' requirements or addressed to gene bank managers' resource constraints. Arguments given in favour of developing core subsets, rather than a single core are: (i) logical; (ii) population genetic; and (iii) germplasm usage considerations. The Indian PGR programme and the emerging core context are briefly discussed, along with features of a programme designed to develop such core subsets.Abbreviations IARCs = International Agriculture Research Centers - NBPGR = National Bureau of Plant Genetic Resources - NAGS = National Active Germplasm Collection Site - NGSNs = National Germplasm Screening Nurseries - GACs = Germplasm Advisory Committees     

Recombinase Polymerase Amplification assay for rapid detection of a geminivirus associated with potato apical leaf curl disease

Journal of Plant Diseases and Protection - Recombinase Polymerase Amplification (RPA) assay was developed for specific detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV [potato]),...     

Conversion of Potato Starch and Peel Waste to High Value Nanocrystals

The present study aimed to convert starch and potato peel waste to nanocrystals. Starch nanocrystals were prepared using two methodologies: direct acid hydrolysis and enzyme pretreatment followed by acid hydrolysis. Direct hydrolysis broke down the starch granules to nanocrystals in 12 days. Enzyme pretreatment with starch hydrolytic enzymes (α-amylase and amyloglucosidase) reduced the time for preparation of starch nanocrystals by 6 days. Starch nanocrystals of optimum size were obtained with both the treatments and the resultant size ranged from 10 to 50 nm. Nanocrystals were disk-like platelets in appearance. Cellulose nanocrystals were derived from cellulosic material in the potato peel. Cellulose was isolated from peel waste with alkali treatment. Further, cellulose nanocrystals from potato peel and cellulose microcrystalline were prepared by acid hydrolysis. Microscopic images revealed that the aqueous suspension of cellulose nanocrystals derived from potato peel were single rod shaped, whereas those derived from cellulose microcrystalline were rod-like nanoparticles, agglomerated in the form of bundles including some of the rods in single units (well separated). The size of potato peel nanocrystals ranged from 40 to 100 nm (length) and cellulose microcrystalline ranged from 4 to 20 nm (diameter) by 110 to 250, given 4 to 20 nm (length), respectively. As starch nanocrystals as well as cellulose nanocrystals are derived from biopolymer, both can be considered safe for humans and the environment. Moreover, the biodegradable nature of these nanocrystals makes them superior over metallic nanoparticles, particularly in the field of nanocomposites.     

Short-chain peptide analysis by high-performance liquid chromatography coupled to electrospray ionization mass spectrometer after derivatization with 9-fluorenylmethyl chloroformate

Resolution and characterization of short-chain peptides (M(r) = 200-1000) and free amino acids were demonstrated by the use of precolumn derivatization with 9-fluorenylmethyl chloroformate (Fmoc) followed by reverse-phase high-performance liquid chromatography (RP-HPLC) interfaced with an electrospray ionization mass spectrometer (ESI-MS). At pH 10, in addition to derivatization at the N terminus, epsilon-NH(2) and OH groups of lysine and tyrosine residues, respectively, were also derivatized. Fmoc derivatives showed at least 2 orders of magnitude higher ionization potential in the presence of trifluoroacetic acid. The detection levels for both the free amino acid and peptide derivatives were in a few hundred picomoles compared to 10-50 nmol for the underivatized samples. The mass spectra of the peptides before or after derivatization showed the presence of only singly charged ions. However, collision-induced dissociation of the derivatized peptides showed predominance of b-type ions that are relatively less complicated in assigning the peptide sequence.     

Production of sexed lambs after biopsy of ovine blastocyts produced in vitro

Embryos were generated by in vitro fertilization of in vitro-matured oocytes, cultured to the blastocyst stage, biopsied for sex determination by a PCR-based procedure, and transferred to synchronized recipients. Three out of 5 sheep (60%) were diagnosed pregnant, and 4 lambs of predicted sex were born.     

Maturation Status, Protein Synthesis and Developmental Competence of Oocytes Derived from Lambs and Ewes

The efficacy of oocyte selection for in vitro embryo production depends on the abundance and diameter of follicles, cumulus layers around the oocytes and subsequent fertilization. Application of `ovum pick-up' technique allows us to utilize partially matured oocytes for embryo production even from juvenile subjects. To compare their developmental competence, oocytes derived from lambs and ewes and cultured in maturation medium for up to 26 h were assessed at 2 h intervals by confocal microscopy after chromatin and microtubulin-specific fluorochrome labelling. Lamb oocytes reached second meiotic metaphase (MII) at lower numbers at 24 h (60.0%) and 26 h (28.6%) whereas 85.7% of adult-derived oocytes attained MII status by 24 h of maturation. Radiolabelling of oocyte proteins revealed higher incorporation of [³⁵S-]-methionine and [³⁵S]-cysteine in adult-derived oocytes compared to lamb oocytes. Although the cleavage rate of lamb oocytes was similar to that of ewe oocytes, the proportion reaching blastocyst stage was significantly lower (p < 0.05) in the lamb-derived oocytes. However, blastocysts from both types of oocytes displayed similar cell lineage allocations to inner cell mass and trophectoderm.     

Carbohydrate Metabolism in the Siliqua Relating to Oil-filling in Mustard Seeds

Developing siliquas on the mustard inflorescence were sampled at basal, middle and apical positions and the changes in free sugars and starch in pod wall and seed vis-á-vis oil-filling in the seeds were studied. The dry matter and oil content per seed and pod wall was highest at initial stages in apical followed by mid-development stages in middle and late development stages in basal positions. The oil percentage m the pod wall decreased with the period of siliqua development. The phase of rapid oil filling in the seeds varied from 20 to 40 DAF (days after flowering) in basal to 10 to 30 DAF in middle and 10 to 20 DAF in apical positions. The content of starch and total soluble sugars (% dry weight basis) decreased in the seeds as well as pod walls but showed accumulation on per seed basis with a maximum at 20, 30 and 40 DAF while on pod wall basis, the maxima of total soluble sugars was at 20, 20 and 40 DAF in apical, middle and basal position respectively. In the pool of total soluble sugars, the proportion of non-reducing sugars was predominant. The activity of invertase (EC 3.2.1.26) declined while those of a-amylase (EC 3.2.1.1) and β-amylase (EC 3.2.1.2) showed maximum values in the seeds as well as pod wall during the phase of rapid oil-filling in the seeds. The results suggested that ontogeny and duration of seed development vis-á-vis the environmental conditions played an important role in lipid biosynthesis in mustard seeds.     

Primary structure of the abundant seed albumin of Theobroma cacao by mass spectrometry

The most abundant albumin present in seeds of Theobroma cacao was purified to apparent homogeneity as judged by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH(2)-terminal sequence analysis. Tryptic peptide mass fingerprinting of the purified protein by HPLC/ESI-MS showed the presence of 16 masses that matched the expected tryptic peptides corresponding to 95% of the translated amino acid sequence from the cDNA of the 21 kDa cocoa albumin. Collision-induced dissociation MS/MS analysis of the C-terminal peptide isolated from the CNBr cleavage products provided unequivocal evidence that the mature cocoa albumin protein is nine amino acid residues shorter than expected from the reported cDNA of its corresponding gene. The experimentally determined M(r) value of 20234 was in excellent agreement with the truncated version of the amino acid sequence. The purified cocoa albumin inhibited the catalytic activities of bovine trypsin and chymotrypsin. The inhibition was stoichiometric with 1 mol of trypsin or chymotrypsin being inhibited by 1 mol of inhibitor with apparent dissociation constants (K(i)) of 9.5 x 10(-8) and 2. 3 x 10(-6) M, respectively, for inhibitor binding at pH 8.5 and 37 degrees C. No inhibition of the catalytic activities of subtilisin, papain, pepsin, and cocoa endoproteases was detected under their optimal reaction conditions.