Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.

Analysis of the activity of promoters from two photosynthesis-related genes psaF and petH of spinach in a monocot plant, rice.

The subunit III of photosystem I and ferredoxin-NADP(+)-oxidoreductase are encoded by nuclear genes, namely psaF and petH. The activity of their promoters from spinach has been evaluated in transgenic tobacco earlier. Evaluation of the activity of these Dicotyledoneae-specific promoters has been carried out in a monocot system (i.e. rice) by transient gene expression system, based on electroporation-mediated gene delivery into protoplasts from leaves and roots. It has been found that various promoter deletions show higher activity in leaf protoplasts and elements for quantitative response are widely distributed. Transgenic rice has also been produced with a petH promoter and gus reporter gene construct. Although petH promoter is a weak promoter in comparison to the 35S promoter, it expresses well in green tissues and could be useful for plant genetic engineering.

Alternative approach and evaluation of three potency assay methods for testing adsorbed tetanus toxoid.

Thirteen batches of adsorbed Tetanus Toxoid (TT) from different manufacturers were tested for potency by three different methods viz: (i) An Antibody Induction Method (AIM) developed in mice: (ii) WHO lethal challenge in mice; and (iii) Conventional Antibody Induction (I.P). Method in guinea pigs. The potency results obtained in AIM, by serological evaluation of immunized mice were found identical and correlated significantly with those obtained by WHO recommended lethal challenge test in mice. The potency data obtained in the present study was found comparable with other studies. An AIM in mice thus offers an alternative to lethal challenge tests and can replace guinea pig model. Out of 107 serum samples obtained from immunized guinea pigs in the conventional antibody induction method, 90% samples contained more than 4 units of tetanus antitoxin per ml. End point titres of 42 serum samples belonging to 5 batches of TT also showed much higher tetanus antitoxin content when determined by TN test. The potency data obtained thus suggest revision of the minimum requirement in Indian Pharmacopoeia which is too low and which may be increased as indicated by the present study.

Quantitation of tetanus and diphtheria antitoxins in mouse sera by indirect haemagglutination.

Serum samples obtained from 75 groups of mice immunized with various doses of adsorbed tetanus vaccine, adsorbed diphtheria-tetanus vaccine and adsorbed diphtheria-tetanus-pertussis vaccine were titrated for tetanus antitoxin content by an in-vitro indirect haemagglutination (IHA) and by toxin neutralization test (TN) in mice. From these serum samples of 49 groups of mice which were immunized with combined vaccine containing diphtheria toxoid were titrated for their diphtheria antitoxin content by IHA and by i.d. toxin neutralization test (TN) in guinea pigs. Good correlations were found between the estimates obtained by in-vitro IHA and in vivo TN tests in both tetanus and diphtheria antitoxin titrations. The minimum level of tetanus or diphtheria antitoxin detectable by IHA was 0.00039 IU/ml. It is concluded that IHA is a simple, sensitive and reproducible alternative test which can replace the animal TN tests for the estimation of tetanus and diphtheria antitoxins and could reliably be used in the potency assay of tetanus and diphtheria toxoids of combined vaccines based on antibody induction in mice.

An antibody induction method in mice for the potency assays of diphtheria and tetanus components in combined vaccines.

Eleven batches of Adsorbed Diphtheria-Tetanus (DT) vaccines and thirteen batches of Adsorbed Diphtheria-Pertussis-Tetanus (DTP) vaccines were tested for the potency of diphtheria and tetanus components by an Antibody Induction Method (AIM) developed in mice. The potency results obtained were found comparable and did not show any statistically significant difference with those obtained by WHO recommended lethal challenge tests for diphtheria in guinea pigs and for tetanus in mice. AIM in mice is more economical as both diphtheria and tetanus components of combined vaccine can be tested in the same experiment and the procedure also eliminates the use of guinea pigs required in the lethal challenge/conventional tests. The data obtained while testing tetanus component by the conventional antibody induction (IP) method in guinea pigs suggests that minimum requirements laid down in i.p. is too low which may be fixed as at least 3 out of 9 guinea pig sera and should contain > or = 4 units of tetanus antitoxin per ml.

Differentiation in explants from mature leguminous trees.

Stem and petiole explants, obtained from mature trees, ofAlbizzia lebbeck,Cassia fistula andC.siamea callused and differentiated shoot-buds and later shoots on B5 medium supplemented with either 0.5 mg/l IAA + 1 mg/l BAP or BM + 2 mg/l NAA + 0.5 mg/l BAP. The stem explants were more responsive than the petiole explants. InA.lebbeck, the IAA substituted medium favoured differentiation from both types of explants. However, inC.fistula, the type of explants rather than the medium composition had an overriding influence on shoot differentiation since those from petiole hardly responded in either medium. It has been possible to obtain plantlets from bothA.lebbeck andC.fistula under conditions conducive to rooting. Plantlets ofA.lebbeck have also been successfully transferred to the field.

Preliminary characterization of a cholinesterase from roots of Bengal gram - Cicer arietinum L.

A Cholinesterase was isolated and purified (65-fold) from roots of Bengal gram (Cicer arietinum L.) seedlings. It hydrolyzes acetylcholine and acetylthiocholine more readily than propionylthiocholine or butyrylthiocholine. The enzyme has high affinity for acetylthiocholine (Km=150 µm) and is inhibited by animal anticholinesterases, neostigmine and physostigmine, and by Phosfon-D, a plant growth retardant.

Genetic and physiological influences on differentiation in tissue cultures of a legume, Lathyrus sativus.

Callus initiation and subsequent organogenetic potential in cultures of shoot meristems of Lathyrus sativus is under genetic control. This genetic influence is observable not only at the broader intercultivar level, but also between genotypes descended from the same cultivar. However, it is possible to achieve higher levels of morphogenetic response even in recalcitrant genotypes by using the physiologically altered explant. On activation of the dormant lateral bud meristems consequent to decapitation of the apical bud, it is possible to induce organogenesis in the tissues of all genotypes. Callus masses arising from the shoot meristems and leaf expiants of cv. 'LSD-3' also exhibit somatic embryogenesis which results in the development of mature plantlets under culture conditions.

Induction of flowering in Lemna paucicostata, a short-day plant, by chelating agents and iron.

Lemna paucicostata is a short-day plant which normally flowers only in a medium supplemented with EDTA or EDDHA. On a molar basis EDDHA is more effective for induction of flowering. The chelating agent can be replaced by high concentrations of ferric citrate in the medium. Simultaneous supply of both EDDHA and a high level of ferric citrate results in flowering even under long days.

Cytokinins in seeds of pumpkin.

Extracts of seeds of pumpkin (Cucurbita pepo Linn.) contain three chromatographically distinguishable cytokinins which are held on Dowex 50-W and are extractable by ethanol and n-butanol. Two of the active factors are precipitable by silver nitrate at acidic pH. The chromatographic behavior and the spectral characteristics of one of these cytokinins are similar to those of zeatin. However, the R(F) values of the other two active compounds do not match with those of any of the known natural cytokinins.

Transient expression of gus gene in intact seed embryos of Indica rice after electroporation-mediated gene delivery.

Two-day-old germinating intact seed embryos of Oryza sativa variety Basmati 370 were electroporated with a view to examine suitability of this system for gene delivery. The experiments were done with a plasmid having gus gene under the control of CaMV 35S promoter. Spectrofluorophotometric GUS assay revealed high activity of the introduced gene when embryos were given three electrical pulses at 1600 V cm(-1) and 100 µF capacitance with a pulse length of 75 ms. Additionally, histochemical localization of GUS activity in seedlings and various organs such as leaves, coleoptiles and roots was also done. Expression of GUS activity was studied up to 15 days and found to be organ-specific, thereby showing that embryos can indeed serve as efficient recipient system. Use of cycloheximide revealed that GUS activity appears as a result of early protein synthesis after electroporation and is substantially stable in vivo.

Developmental and light-dependent cues interact to establish steady-state levels of transcripts for photosynthesis-related genes (psbA, psbD, psaA and rbcL) in rice (Oryza sativa L.).

The steady-state transcript levels for psbA, psbD, psaA and rbcL are low in dark-grown rice seedlings as compared to those grown in light. Following seed germination, they accumulate in an age-dependent manner, in dark as well as light, reaching a maximal level on the 7th or 8th day, before a slow decline sets in. But transcripts for psbA and psbD continue to maintain relatively-high levels even after 10 days of growth in light. Exposure of 5-day-old dark-grown seedlings to light results in an approximately 25-60-fold increase in transcripts during a period of 72 h, followed by a decrease. An analysis of data from both lines of investigation reveals that the developmental programme increases the transcript levels for psbA, psbD, psaA and rbcL by about 10-, 2.3-, 7.0- and 8.0-fold, respectively, between 5-8 days after germination and it is independent of light. At the same time, exposure of the seedlings to light during this period further enhances transcript levels by 5-, 11.4-, 6.6- and 7.8-fold, respectively. Thus, both developmental and light-dependent cues contribute to establish steady-state levels of transcripts for the chloroplast genes investigated.