Upstream sequence of fatty acyl-CoA reductase (FAR6) of Arabidopsis thaliana drives wound-inducible and stem-specific expression.

An Arabidopsis mutant line T90, exhibiting a stem-specific and wound-responsive GUS expression was identified from a population of Arabidopsis thaliana tagged with a promoterless ß-glucuronidase (GUS) in the T-DNA. Sequence flanking the insertion from the right border was amplified by TAIL PCR and cloned. The insertion was located in the third chromosome, 57 bp upstream of the ATG start codon in 5' untranslated region (UTR) of the fatty acyl-CoA reductase 6 (FAR6) gene. RT-PCR analysis of the FAR6 gene revealed that the gene is expressed predominantly in stem tissue. Semi-quantitative RT-PCR showed that the expression is also induced by wounding in the epidermal layer of mature stem internodes. The transcription initiation site (TSS) was identified by 5' RACE PCR. Different 5' deletion fragments of the promoter sequences were developed and linked to the GUS reporter gene as transcriptional fusions and the expression patterns of GUS were histochemically analyzed in transgenic Arabidopsis plants. Sequences from -510 bp upstream to the transcriptional start site were sufficient to exhibit wound-inducible GUS expression in the stems. The addition of further upstream sequences (-510 to -958, -1,400 or -1,456) enhanced and extended the wound-inducible GUS expression throughout the mature stem.

A duplicated coxI gene is associated with cytoplasmic male sterility in an alloplasmic Brassica juncea line derived from somatic hybridization with Diplotaxis catholica.

A cytoplasmic male sterile (CMS) line of Brassica juncea was derived by repeated backcrossing of the somatic hybrid (Diplotaxis catholica + B. juncea) to B. juncea. The new CMS line is comparable to euplasmic lines for almost all characters, except for flowers which bear slender, needle-like anthers with aborted pollen. Detailed Southern analysis revealed two copies of coxI gene in the CMS line. One copy, coxI-1 is similar to the coxI gene of B. juncea, whereas the second copy, coxI-2 is present in a novel rearranged region. Northern analysis with eight mitochondrial gene probes showed altered transcript pattern only for the coxI gene. Two transcripts of 2.0 and 2.4 kb, respectively, were detected in the CMS line. The novel 2.4 kb transcript was present in floral bud tissue but absent in the leaf tissue. In plants where male sterility broke down under high temperature during the later part of the growing season, the 2.4 kb coxI transcript was absent, which suggested its association with the CMS. The two coxI genes from the CMS line showed two amino acid changes in the coding region. The novel coxI gene showed unique repeats in the 5' region suggesting recombination of mitochondrial genomes of the two species. The possible role of the duplicated coxI gene in causing male sterility is discussed.

Identification of AFLP markers linked to the male fertility restorer gene of CMS (Moricandia arvensis) Brassica juncea and conversion to SCAR marker.

We have developed a cytoplasmic male sterile (CMS) line of Brassica juncea through somatic hybridization with Moricandia arvensis and introgressed the fertility restorer gene into B. juncea. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. As a first step toward cloning of this restorer gene we attempted molecular tagging of the Rf locus using the amplified fragment length polymorphism (AFLP) technique. A BC(1)F(1) population segregating for male sterility/fertility was used for tagging using the bulk segregant analysis method. Out of 64 primer combinations tested in the bulks, 5 combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons mostly in the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM. All the markers are located on one side of the Rf locus.

Homeotic-like modification of stamens to petals is associated with aberrant mitochondrial gene expression in cytoplasmic male sterile Ogura Brassica juncea.

We have previously reported correction of severe leaf chlorosis in the cytoplasmic male sterile Ogura (also called Ogu) Brassica juncea line carrying Ogura cytoplasm by plastid substitution via protoplast fusion. Two cybrids obtained from the fusion experiment, Og1 and Og2, were green and carried the plastid genome of B. juncea cv. RLM198. While Og1 displayed normal flower morphology comparable to that of its euplasmic B. juncea counterpart except for sterile anthers, Og2 retained homeotic-like floral modification of stamens to petal-like structures and several other floral deformities observed in the chlorotic (Ogu) B. juncea cv. RLM198 (or OgRLM). With respect to the mitochondrial genome, Og1 showed 81% genetic similarity to the fertile cultivar RLM while Og2 showed 93% similarity to OgRLM. In spite of recombination and rearrangements in the mitochondrial genomes in the cybrids, expression patterns of 10 out of 11 mitochondrial genes were similar in all the three CMS lines; the only exception was atp6, whose expression was altered. While Og1 showed normal atp6 transcript similar to that in RLM, in Og2 and OgRLM weak expression of a longer transcript was detected. These results suggest that the homeotic-like changes in floral patterning leading to petaloid stamens in Og2 and OgRLM may be associated with aberrant mitochondrial gene expression.

T-DNA tagging and characterization of a cryptic root-specific promoter in Arabidopsis.

From a T-DNA tagged Arabidopsis population, a line, M-57 showing GUS (beta-glucuronidase) expression in the vascular regions of young roots was identified. Southern analysis revealed presence of a single T-DNA insert. Using inverse PCR, the plant sequence flanking the T-DNA insertion was cloned. The insertion was identified to be in the intergenic area between loci At4G13940 and At4G13930, coding for SAHH (S-Adenosyl-l-Homocysteine Hydrolase) and SHMT (Serine Hydroxy Methyl Transferase) genes, respectively. A 452-bp fragment immediately upstream of the T-DNA insertion when cloned and mobilized as a GUS fusion was capable of driving a similar root-specific expression of reporter gene in transgenic Arabidopsis plants and their progenies. This cryptic promoter element does not show the presence of any known root-specific promoter element.

Cloning and characterization of a pentatricopeptide protein encoding gene (LOJ) that is specifically expressed in lateral organ junctions in Arabidopsis thaliana.

A line exhibiting expression of beta-glucuronidase (GUS) in the lateral organ junctions and shoot apical meristem (SAM) was identified from a population of T-DNA tagged lines carrying a promoter-less GUS gene. Southern hybridization confirmed the presence of a single T-DNA insertion in this line. The plant sequences flanking the T-DNA were cloned by TAIL PCR and sequenced. The insertion of T-DNA was found to be in the upstream region of a hypothetical gene (At2g39230). This gene, which we term as LOJ to indicate its specific expression in all lateral organ junctions encodes a predicted protein containing pentatricopeptide (PPR) motifs. This gene appears to belong to a group of TATA-less promoters and codes for a long ORF without any intron. The gene apparently codes for a protein of 97.65 kD with a mitochondrial target sequence at the N-terminal. Transcript analysis revealed that the expression of the gene is specifically restricted to the lateral organ junctions throughout the life of the plants. 5' RACE analysis revealed a 95 nucleotide long UTR region for this hypothetical gene. In silico analysis of the upstream region failed to identify a TATA box within -146 nucleotides. GUS expression analysis of the line 149 and the transgenic plants generated with constructs carrying the upstream sequences of this gene fused to uidA identified that the specificity of the expression of this gene resides within -569 to -152 bp region. The specific expression of LOJ at the base of lateral organ and shoot apical meristem (SAM) suggests an important role of LOJ in lateral organ development and boundary demarcation.

Cytoplasmic male sterility in alloplasmic Brassica juncea carrying Diplotaxis catholica cytoplasm: molecular characterization and genetics of fertility restoration.

The present study was aimed at characterizing cytoplasmic male sterility (CMS) and identifying the fertility restorer gene for CMS (Diplotaxis catholica) Brassica juncea derived through sexual hybridization. The fertility restorer gene was identified by crossing the CMS line with progeny plants derived from somatic hybrids of B. juncea and D. cathoilca. The CMS line is comparable to the nuclear donor B. juncea in all respects except for flower and silique characteristics. In CMS plants, the flowers have smaller nectaries, and anthers are converted into petals or tubular structures. Gynoecium exhibits a crooked style and trilocular ovary. Seed fertility was reduced in the CMS line. Genetic segregation data indicated that a single, dominant, nuclear gene governs fertility restoration. Restored plants showed a high female fertility and lacked gynoecium abnormalities. In fertility-restored plants, petal development was found to be variable; some flowers had the normal number of four petals, while others had zero to three petals. Interestingly, the trilocular character of the ovary was found to co-segregate with CMS and became bilocular upon male-fertility restoration. Thus, this trait appears to be affected by the interaction of nuclear and mitochondrial (mt) genomes. Restriction fragment length polymorphism analysis indicated that mt-genome of D. catholica is highly divergent from that of B. juncea. However, in Northern analysis, out of eight mt genes studied, an altered transcript pattern was recorded for only atpA. In fertility-restored plants, the atpA transcript became shorter, thereby showing its association with CMS.

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower.

A number of factors that are known to influence genetic transformation were evaluated to optimize Agrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized into Agrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the synthetic cryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae

Multicenter randomized placebo controlled trial of therapy with intravenous immunoglobulin in decreasing mortality due to neonatal sepsis.

OBJECTIVE: To determine whether therapy with intravenous immunoglobulin G (IVIG) would decrease mortality in neonatal sepsis. SETTING: Three tertiary care neonatal intensive care units in the city of Bangalore. METHODS: All neonates admitted to the Neonatal Intensive Care Units with the clinical diagnosis of sepsis and having at least C-reactive protein and one other rapid diagnostic criteria positive were enrolled. Neonates with a birth weight of less than 1000 g and those with any major congenital malformation were excluded. The neonates were randomized to receive 1 g/kg of IVIG on three consecutive days or an equivalent amount of placebo. The rest of the treatment including antibiotics and supportive care was as per the treating physician's decision. The main outcome variable was survival. RESULTS: The trial was carried out over a period of 8 months and recruited 58 neonates. Seven neonates who qualified but did not receive either IVIG or placebo were taken into a separate control group, and one baby who received only one dose of IVIG was excluded from the analysis. Twenty-five neonates were enrolled into the IVIG arm and 25 in the placebo arm. The neonates in the therapy and placebo groups were comparable in terms of birth weight (2144+/-675 g vs. 2072+/-682 g), gestation (37.0+/-3.56 vs. 35.8+/-3.52 weeks), sex distribution, duration of stay, and number requiring ventilation. The placebo group had a significantly higher number of babies with positive blood culture. Seven babies in each group died (p>0.05). There was no significant benefit in using IVIG (OR 1.0; 95% CI 0.25-4.07) (p = 0.74). CONCLUSION: In the sample studied therapy with IVIG did not reduce mortality in neonatal sepsis

DNA fingerprinting of Musa cultivars with oligodeoxyribonucleotide probes specific for simple repeat motifs.

Using synthetic oligodeoxyribonucleotide probes against restriction-digested genomic DNA, we have established DNA fingerprinting of Musa cultivars. Of all the enzymes used, Eco RI and Hin dIII were found to be most informative, giving rise to individual specific band patterns with oligonucleotide probes of 15- to 18-base residues. Of the several probes and enzyme combinations used, the 15mer GACA probe with Eco RI and Hin dIII digests revealed a maximal level of polymorphism, and the probability of obtaining an identical band pattern between any two random genotypes was calculated to be 1.50 x 10(-9) and 1.59 x 10(-9), respectively. Oligonucleotide probes longer than 22 residues were also used but did not hybridize. The present approach is useful for cultivar identification and for overall genome analysis to establish relatedness among the various accessions of the Musa germplasm originating from different geographic locations. The relevance of using synthetic oligonucleotide probes based on simple repeat motifs for achieving DNA fingerprinting pattern is discussed.

Plant regeneration from various expiants of cultivated Piper species.

Morphogenetic potential of root, leaf, node and internode expiants of 3 cultivated Piper species was investigated to develop a reliable plant regeneration protocol. P. longum (pipli) was the most responsive followed by P. betle (betel vine) and P. nigrum (black pepper). In P. longum the highest number of shoot buds was produced on root expiants followed by node, internode and leaf expiants. In P. betle and P. nigrum adventitious shoot buds differentiated only from internodal and nodal ring regions, respectively. Histological examination in P. longum showed that adventitious shoot buds originate directly from the cortical cells of the root and the internode without an intervening callus phase. Benzyladenine was superior to kinetin for shoot induction and its optimum concentrations for P. longum, P. betle and P. nigrum were 1-2, 10 and 10 µM, respectively. Shoot elongation and rooting were achieved in B5 medium containing 0.5 µM benzyladenine and 1 µM indoleacetic acid, respectively. Regenerated plants were established in soil.

Plant regeneration from callus cultures of Piper longum L. by organogenesis.

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l(-1)α- naphthaleneacetic acid and 0.2 mg.l(-1) N(6)-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l(-1) indole-3-acetic acid with 1.5 mg.l(-1) benzyladenine, and 0.1 mg.l(-1) indole-3-acetic acid with 0.2 mg.l(-1) benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l(-1) indole3-acetic acid. The rooted plants were successfully established in soil.

A novel technique to overcome browning in tissue culture.

Experiments conducted using Dioscorea alata L. revealed that an exudate from the cut end of the explants was responsible for browning of the culture medium. Browning did not affect growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Sealing the cut ends with paraffin wax was found to control browning by preventing exudation. This simple technique permitted establishment of cultures in a small volume of medium in about 90 percent of the cases, while in unsealed cultures lethal browning was recorded in 80 percent of the cases. The advantages of this technique over other methods of controlling browning are discussed.

In vitro clonal multiplication of turmeric (Curcuma spp.) and ginger (Zingiber officinale Rosc.).

Rhizome buds, excised from threeCurcuma spp., and ginger, inoculated aseptically on MS medium with varying levels of BAP and kinetin, produced multiple shoots. For shoot multiplication, a concentration of 3.0 mg/l BAP was found to be optimum for all the species.In vitro plants were successfully established in the field and were morphologically uniform. A simple method to extend the subculture interval was used and its relevance to germplasm conservation is discussed.

Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets.

Conditions necessary for the isolation and culture of protoplasts from suspension cultures of sugar, fodder and garden beets were investigated. Good yields of protoplasts were obtained by treating cells with a mixture of cellulase, Macerozyme and Driselase enzymes. Nutritional requirements of beet protoplasts were found to be quite simple: protoplasts could be cultured in MS, B5 or PGo based media with 0.4 M glucose with the optimum result being produced on KM8p medium. Plating efficiency (P.E) was genotype-dependent with the sugar beet giving better P.E. than the fodder or garden beets used, and higher values being achieved with the use of desalted Driselase for isolation followed by culture on KMBp medium.