Embryo sac cellularization defects lead to supernumerary egg cells and twin embryos in Arabidopsis thaliana.

Arabidopsis lines with loss-of-function mutation in Embryo sac-specific Pectin MethylEsterase Inhibitor (Atepmei) gene showed seed sterility with embryo sac cellularization defects. Examination of tissue-cleared mature ovules revealed irregularly positioned nuclei/embryos within the embryo sacs. Egg cell-specific marker (DD45) expression analysis confirmed the presence of multiple egg cells in the mutant embryo sacs. These supernumerary egg cells were functional as evident from the production of twin embryos when supernumerary sperm cells were provided. The results of ruthenium red and tannic acid-ferric chloride staining of developing Atepmei mutant ovules showed that cell wall formation and maintenance were altered around embryo sac nuclei, which also coincided with change in the gamete specification. This report implicates the role of cell walls in gamete cell fate determination by altering cell-cell communication. Our analysis of the twin-embryo phenotype of epmei mutants also sheds light on the boundary conditions for double fertilization in plant reproduction.

Analysis of mitochondrial recombination in the male sterile Brassica juncea cybrid Og1 and identification of the molecular basis of fertility reversion.

KEY MESSAGE: Recombinations between the parental genomes produced a novel mitochondrial genome in the cytoplasmic male sterile Brassica juncea cybrid Og1. A mitochondrial stoichiometric shift greatly reduced the molecule containing male-sterility-inducing orf138 gene leading to reversion to male fertility. An improved, chlorosis-corrected, cytoplasmic male sterile Brassica juncea cybrid Og1 derived from Ogura cytoplasm shows frequent reversion to male fertility. To determine the nature of mitochondrial recombination in the cybrid and to uncover the molecular mechanism of male fertility reversion, we sequenced the mitochondrial genomes of Og1, its isonuclear parental lines (OgRLM and Brassica juncea RLM198) and the revertant line (Og1-rt). Assembly of Og1 mitochondrial genome gave two circular molecules, Og1a (250.999 kbp) and Og1b (96.185 kbp) sharing two large direct repeat regions capable of recombining to form a single circular molecule. Og1a contains all essential mitochondrial genes, but the male-sterility-causing orf138 was uniquely present in Og1b along with 16 other complete or partial genes already represented in Og1a. Eleven and four recombinations between the parental mitochondrial genomes produced the Og1a and the Og1b molecules, respectively. Five genes were duplicated within Og1a, of which trnfM was inherited from both the parents while the other four genes, atp4, cox1 nad4L and trnM, were inherited from RLM198. RFLP analysis revealed that orf138-containing molecules were less abundant than Og1a in the male-sterile plants while og1b bearing molecules were undetectable in the revertant line. However, orf138 transcripts were amplified in RT-PCR and were also detected in northern blots revealing that Og1b molecules are not completely lost in the revertant plants. This is the first report where the mitochondrial genome of a cybrid is compared with its actual parents. The findings are discussed in the light of previous reports on mitochondrial genome recombination in cybrids.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants.

A promoter trap mutant line of Arabidopsis carrying a promoterless ß-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development.

In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants.

Analysis of Promoters of Arabidopsis thaliana Divergent Gene Pair SERAT3;2 and IDH-III Shows SERAT3;2 Promoter is Nested Within the IDH-III Promoter.

Intergenic regions of divergent gene pairs show bidirectional promoter activity but whether regulatory sequences for gene expression in opposite directions are shared is not established. In this study, promoters of divergently arranged gene pair At4g35640-At4g35650 (SERAT3;2-IDH-III) of Arabidopsis thaliana were analyzed to identify overlapping regulatory regions. Both genes showed the highest expression in flower buds and flowers. 5' RACE experiments extended the intergenic region from 161 bp shown in TAIR annotation to 512 bp. GUS analysis of transgenic A. thaliana plants carrying the 691 bp fragment (512 bp intergenic region plus 5' UTR of both the genes) linked to uidA gene revealed that SERAT3;2 promoter drives gene expression in the tapetum, whereas IDH-III promoter functions specifically in microspores/pollen. Serial 5' deletion of the 691 bp fragment showed SERAT3;2 promoter extends up to -355 position, whereas IDH-III promoter encompasses the 512 bp intergenic region. In transgenics, uidA transcript levels were lower than native SERAT3;2 and IDH-III transcripts indicating presence of additional cis regulatory elements beyond the 691 bp fragment. The present study demonstrated for the first time occurrence of a nested promoter in plants and identified a novel bidirectional promoter capable of driving gene expression in tapetum and microspores/pollen.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation.

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at -309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5' UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300 bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.

Assessment of Arabidopsis thaliana CENH3 promoter in Brassica juncea for development of haploid inducer lines.

Centromeres are epigenetically specified by the centromeric histone H3 protein (CENH3). The timing and level of expression of CENH3 is tightly regulated to match the demands of the host cell. So far in plants, only CENH3 promoter of Arabidopsis thaliana (L.) Heynh. has been characterized. However, whether CENH3 promoters retain their characteristic mode of regulation in other species remains to be established. In the present study, activity of AtCENH3 promoter was investigated using reporter gene assay in Brassica juncea (L.) Czem. A 1156 bp promoter fragment of AtCENH3 gene (At1g01370) including the first 111 nucleotides of the coding sequence was amplified and cloned into the pORE-R2 binary vector to ensure translation fusion with the uidA coding sequences. The Agrobacteriun tiunefaciens strain GV3101 harbouring the recombinant construct was used to transform B. juncea cv. RLM198 hypocotyl explants. Histochemical assay of To and T, transgenics showed GUS expression in shoot apical meristem, leaf, sepal, flower pedicel and root tip. Intense GUS expression was observed in meristematic tissues, particularly at shoot and root apices. However, mature leaves, flowers, pollen and ovules exhibited very low or no GUS expression. Our results showed that AtCENH3 promoter regulates cognate gene expression in Brassica juncea as it does in A. thaliana, and hence a suitable candidate for developing haploid inducer line in B. juncea.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.).

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.

Cytoplasmic male sterility in Brassicaceae crops.

Brassicaceae crops display strong hybrid vigor, and have long been subject to F1 hybrid breeding. Because the most reliable system of F1 seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.

Non-codified traditional medicine practices from Belgaum Region in Southern India: present scenario.

BACKGROUND: Traditional medicine in India can be classified into codified (Ayurveda, Unani, Siddha, Homeopathy) and non-codified (folk medicine) systems. Both the systems contributing equally to the primary healthcare in India. The present study is aimed to understand the current scenario of medicinal practices of non-codified system of traditional medicine in Belgaum region, India. METHODS: The study has been conducted as a basic survey of identified non-codified traditional practitioners by convenience sampling with semi structured, open ended interviews and discussions. The learning process, disease diagnosis, treatment, remuneration, sharing of knowledge and socio-demographic data was collected, analysed and discussed. RESULTS: One hundred and forty traditional practitioners were identified and interviewed for the present study. These practitioners are locally known as "Vaidya". The study revealed that the non-codified healthcare tradition is practiced mainly by elderly persons in the age group of 61 years and above (40%). 73% of the practitioners learnt the tradition from their forefathers, and 19% of practitioners developed their own practices through experimentation, reading and learning. 20% of the practitioners follow distinctive "Nadi Pariksha" (pulse examination) for disease diagnosis, while others follow bodily symptoms and complaints. 29% of the traditional practitioners do not charge anything, while 59% practitioners receive money as remuneration.Plant and animal materials are used as sources of medicines, with a variety of preparation methods. The preference ranking test revealed higher education and migration from villages are the main reasons for decreasing interest amongst the younger generation, while deforestation emerged as the main cause of medicinal plants depletion. CONCLUSION: Patrilineal transfer of the knowledge to younger generation was observed in Belgaum region. The observed resemblance in disease diagnosis, plant collection and processing between non-codified traditional system of medicine and Ayurveda require further methodical studies to establish the relationship between the two on a more objective basis. However, the practice appears to be at crossroads with threat of extinction, because of non-inheritance of the knowledge and non-availability of medicinal plants. Hence conservation strategies for both knowledge and resources at societal, scientific and legislative levels are urgently required to preserve the traditional wisdom.

Characterization of a T-DNA promoter trap line of Arabidopsis thaliana uncovers a cryptic bi-directional promoter.

Investigation of the transgenic Arabidopsis promoter trap line GFP-868 that showed GFP expression only in anthers revealed the T-DNA insertion at 461bp upstream to the hypothetical gene At4g10596 with the GFP reporter gene in head-to-head orientation to the At4g10596 gene. The expression of the At4g10596 gene in wild type and in GFP-868 plant homozygous for T-DNA insertion was comparable and found in all tissues tested, while the GFP expression was restricted to anthers of the GFP-868 plants suggesting that the 461bp fragment separating the two genes in the GFP-868 line is functioning as bi-directional promoter. This 461bp fragment was cloned upstream to the GUS gene in two orientations to test for bi-directional promoter activity. Transgenic Arabidopsis plants carrying either of these constructs showed GUS activity in anthers indicating that this fragment behaves as bi-directional promoter specific to anthers. These results were also supported by the presence of cis-acting motifs such as TATA box and POLLEN1LELAT52 (AGAAA) within the 461bp sequence in both orientations. However, transcripts corresponding to the upstream sequences beyond -461 nucleotides were not detected in the wild type suggesting that this 461bp fragment is a cryptic promoter. The significance of the promoter trap approach and the usefulness of this type of promoter are discussed.

Ethnomedicinal plants used to treat bone fracture from North-Central Western Ghats of India.

ETHNOPHARMACOLOGICAL RELEVANCE: North Central Western Ghats in India comprises rich bio-cultural diversity and is also home to varied ethnomedicinal practices. The study was taken up for documentation and analysis of traditional knowledge regarding the practice and use of plants in the treatment of bone fracture. This is an effort to preserve the vanishing wealth of traditional knowledge. MATERIALS AND METHODS: Key informants identified in a preliminary survey and collection of information was through semi structured, open ended interviews. The details on age, place of practice, experience of key informants and learning of practice, disease they treat and mode of diagnosis, storage and usage of plants were collected. The identity of plants and their information was confirmed through repeated guided transect walks in different seasons with the informants and focus group discussions. Identified plants were deposited at the herbaria of Regional Medical Research Centre, Belgaum. RESULTS: Forty four key informants providing treatment for bone fracture in this region were identified. Thirty eight plant species belonging to 24 families have been documented in the present study. Highest number of species representation is found in families Euphorbiaceae and Fabaceae. The habit of the species showed that 45% of the herbal drugs were obtained from trees, followed by herbs, shrubs and climbers and majority of plants used were collected from wild (76%). The stem or stem bark (33%) was most commonly used plant part to prepare medicine. Twenty six formulations of 30 plant species were directly used in treating bone fracture, where Cissus quadrangularis has the highest use-value (0.14). Eleven plants were found to be administered for bone strengthening, pain relieving, inflammation reduction and speedy recovery and Gmelina arborea has the highest use value (0.27). CONCLUSIONS: The results indicated the importance of traditional herbal practices in community for their health needs. Both conservational strategies and further validation studies are the need of the hour for better utilization and sustenance of the documented knowledge.

An evolutionarily conserved mitochondrial orf108 is associated with cytoplasmic male sterility in different alloplasmic lines of Brassica juncea and induces male sterility in transgenic Arabidopsis thaliana.

Nuclear-mitochondrial gene interactions governing cytoplasmic male sterility (CMS) in angiosperms have been found to be unique to each system. Fertility restoration of three diverse alloplasmic CMS lines of Brassica juncea by a line carrying the fertility-restorer gene introgressed from Moricandia arvensis prompted this investigation to examine the molecular basis of CMS in these lines. Since previous studies had found altered atpA transcription associated with CMS in these lines, the atpA genes and transcripts of CMS, fertility-restored, and euplasmic lines were cloned and compared. atpA coding and downstream sequences were conserved among CMS and euplasmic lines but major differences were found in the 5' flanking sequences of atpA. A unique open reading frame (ORF), orf108, co-transcribed with atpA, was found in male sterile flowers of CMS lines carrying mitochondrial genomes of Diplotaxis berthautii, D. catholica, or D. erucoides. In presence of the restorer gene, the bicistronic orf108-atpA transcript was cleaved within orf108 to yield a monocistronic atpA transcript. Transgenic expression of orf108 with anther-specific Atprx18 promoter in Arabidopsis thaliana gave 50% pollen sterility, indicating that Orf108 is lethal at the gametophytic stage. Further, lack of transmission of orf108 to the progeny showed for the first time that mitochondrial ORFs could also cause female sterility. orf108 was found to be widely distributed among wild relatives of Brassica, indicating its ancient origin. This is the first report that shows that CMS lines of different origin and morphology could share common molecular basis. The gametic lethality of Orf108 offers a novel opportunity for transgene containment.

Association of AFLP and SSR markers with agronomic and fibre quality traits in Gossypium hirsutum L.

Molecular markers linked to QTL contributing to agronomic and fibre quality traits would be useful for cotton improvement. We have attempted to tag yield and fibre quality traits with AFLP and SSR markers using F(2) and F(3) populations of a cross between two Gossypium hirsutum varieties, PS56-4 and RS2013. Out of 50 AFLP primer combinations and 177 SSR primer pairs tested, 32 AFLP and four SSR primers were chosen for genotyping F(2) individuals. Marker-trait associations were studied for eight agronomic and five fibre quality traits through simple and multiple regression analysis (MRA) using a set of 92 AFLP polymorphic loci and four SSR markers. Simple linear regression analysis (SLRA) identified 23 markers for eight different traits whereas multiple regression analysis identified 30 markers for at least one of the 13 traits. SSR marker BNL 3502 was consistently identified to be associated with fibre strength. While all the markers identified in SLRA were also detected in MRA, as many as 16 of the 30 markers were identified to be associated with respective traits in both F2 and F3 generations. The markers explained up to 41 per cent of phenotypic variation for individual traits. A number of markers were found to be associated with multiple traits suggesting clustering of QTLs for fibre quality traits in cotton.

A novel orf108 co-transcribed with the atpA gene is associated with cytoplasmic male sterility in Brassica juncea carrying Moricandia arvensis cytoplasm.

Mitochondrial atpA transcripts were examined in cytoplasmic male sterile (CMS) and fertility restorer lines of CMS (Moricandia arvensis) Brassica juncea. Male sterile flowers had longer atpA transcripts than male fertiles. The mitochondrial atpA region of the CMS line was cloned and sequenced. The 5' and 3' ends of the atpA transcripts of the CMS and the fertility restorer lines were mapped and full-length transcripts were cloned and sequenced. A novel orf108 (open reading frame 108) co-transcribed with the atpA gene was found in the male sterile flowers. In the fertility restorer line, the transcript was cleaved within orf108 to yield monocistronic atpA transcripts.

Surveillance of drug resistance in tuberculosis in two districts of South India.

SETTING: Tuberculosis Research Centre, Chennai, India. OBJECTIVES: To determine the levels of drug resistance in new and previously treated cases of pulmonary tuberculosis in the composite districts of North Arcot (Tamil Nadu State) and Raichur (Karnataka State) in South India. DESIGN: Two specimens of sputum from 320 patients attending 23 participating centres in North Arcot district and 314 patients from 20 participating centres in Raichur district were tested for drug susceptibility to isoniazid (H), rifampicin (R), ethambutol (E), streptomycin (S) and ofloxacin (O). The studies were undertaken using the guidelines prescribed by the WHO/IUATLD Working Group on Anti-tuberculosis Drug Resistance Surveillance. RESULTS: In North Arcot district, resistance to any drug tested was found in 27.7% of new cases; any H resistance in 23.4%, any R resistance in 2.8% and multidrug resistance (resistance to at least H and R: MDR) in 2.8%. In previously treated cases, resistance to any drug was observed in 81.2%, and any resistance to H, R and HR in 81%, 69% and 69%, respectively. In Raichur district, resistance to any drug was observed in 21.9% of new cases; any resistance to H, R and HR (MDR) was found in 18.7%, 2.5% and 2.5%, respectively. All previously treated patients were resistant to H and R (100%). CONCLUSION: In North Arcot district, the proportion of MDR-TB in newly diagnosed patients has marginally increased over the last 10-15 years, whereas it has remained fairly constant in Raichur district. An increase in resistance was noted in previously treated cases in both districts, although the numbers of patients are small.