Genetic diversity and population structure assessment using molecular markers and SPAR approach in Illicium griffithii, a medicinally important endangered species of Northeast India.

BACKGROUND: Illicium griffithii is an aromatic medicinal tree species that has been listed in the IUCN Red List as an endangered species. Dried seed pods of I. griffithii have a good market potential in the spices and pharmaceutical industries. Fruits are the potential source of shikimic acid and used for the production of oseltamivir (a drug against bird flu). However, in recent years, unscientific harvesting and rampant exploitation of the species has caused a negative and adverse effect on its natural population. Proper knowledge of genetic diversity and population structure is crucial to understand the population dynamics, adaptation, and evolutionary pattern of a particular species for conservation. It was from this view point that the present study was undertaken so as to compare the various types of DNA-based molecular markers namely RAPD, ISSR, DAMD, and SCoT by their efficiency and SPAR approach to evaluate the genetic diversity of I. griffithii as well as to analyze population genetic structure for conservation purpose. RESULT: A total of 250 discernible bands were generated with 246 bands (98.40 %) being polymorphic in nature. All the primers in combination gave a mean polymorphic information content (PIC) of 0.81 and Rp value (resolving power) of 4.32. Nei's, Gst, and AMOVA analysis showed similar values of genetic differentiation among populations (Gst = 0.396, FST = 0.30, respectively), revealing a low level of genetic differentiation among the eight sampled populations. I. griffithii with an estimated gene flow value of Nm = 0.761 was significantly low among populations. Clustering pattern obtained with Bayesian structure and PCoA diagram revealed that intermixing of genetic material across populations is only possible when the populations lie close to each other. This is further validated with UPGMA clustering method where a positive correlation of genetic variability with geographical distance among closely related populations could be clearly seen. CONCLUSION: The result aids in the identification, collection, and preservation of diverse germplasm of I. griffithii from Arunachal Pradesh and Meghalaya of Northeast India. This would further help in understanding the population structure and genetic diversity among other Illicium species in order to formulate effective conservation strategies for the improvement of this endangered taxa.

Induction and evaluation of colchitetraploids of two species of Tinospora Miers, 1851.

Autotetraploidy, both natural and/or induced, has potential for genetic improvement of various crop species including that of medicinal importance. Tinospora cordifolia (Willdenow, 1806) Miers, 1851 ex Hooker et Thomson, 1855 and T. sinensis (Loureiro, 1790) Merrill, 1934 are two diploid species, which are dioecious, deciduous and climbing shrubs with high medicinal importance. Among the three methods used for induction of polyploidy by colchicine treatment, it was cotton swab method which successfully induced the polyploidy in both species. The morphological and cytogenetical features of the synthetic tetraploids were compared with their diploid counterparts. The tetraploids were morphologically distinct from diploid plants. They exhibited larger organs, such as stem, leaves, inflorescence, fruits, flowers and seeds. The tetraploids were characterized by the presence of low quadrivalent frequency and high bivalent average. Unequal distribution of chromosomes at anaphase I was found in 60% cells. The present study provides important information on the superiority of autotetraploids as compared to diploid counterparts in both species.

Molecular strategies for identification and characterization of some wild edible mushrooms of Nagaland, India.

Nagaland has a rich macro fungal flora but not many works has been carried out till today. Present investigation deals with molecular characterization and phylogenetic analysis of six popular wild edible mushrooms (WEMs) species of Nagaland, India viz., Lentinula edodes, Lentinus squarrosulus, L. sajor-caju, L. tigrinus, Schizophyllum commune, Termitomyces heimii and one variety of L. squarrosulus based on molecular markers (ITS, 18S rRNA and 28S rRNA genes) data. The use of DNA markers for identification of mushrooms is highly desirable and practical because it is reliable and quick. This approach could resolve successfully the identity and interrelationship of six WEM species with respect to their infrageneric groups. The high CI values of the mushrooms species indicated the low homoplasy nature. The ITS and 28S rRNA data sets were found to be more informative then the 18S rRNA datasets. The molecular data generated for each mushroom species in the present investigation will help in correct identification and conservation of these widely consumed WEM of the region. Additionally assessment of bioactive molecules indicates that studied species are rich in pro-health bioactive compounds. The study hence throws light on the potential and importance of mushrooms especially the edible mushrooms as an economically valuable crop.

Meiotic behaviour and its implication on species inter-relationship in the genus Curcuma (Linnaeus, 1753) (Zingiberaceae).

In this paper, detailed meiotic analysis was investigated in seven species of Curcuma (Linnaeus, 1753) which can contribute significantly to our understanding about species inter-relationship, speciation and evolution. The species were divided into two groups viz., Group I having 2n = 42 (C. comosa Roxburgh, 1810, C. haritha Mangaly & M.Sabu, 1993, C. mangga Valeton & Zijp, 1917, and C. motana Roxburgh, 1800) and Group II with 2n = 63 (C. caesia Roxburgh, 1810, C. longa Linnaeus, 1753 and C. sylvatica Valeton, 1918). Both groups display varying degree of chromosome associations. Group I species showed the prevalence of bivalents, however occasional quadrivalents besides univalents were also encountered. About 48% of the PMCs analyzed in C. mangga showed 21 bivalents (II) meiotic configurations, 32% in C. comosa and 16% in C. haritha. Group II species as expected showed the presence of trivalents besides bivalents, univalents and quadrivalents. About 32% of the PMCs analyzed at MI in C. sylvatica showed 21 trivalents (III) meiotic configurations, 24% in C. longa and 8% in C. caesia. Overall, low frequency of multivalent associations as compared to bivalents indicates that Curcuma is an allopolyploid complex. Moreover, x = 21 is too high a basic number, therefore, we suggest that the genus Curcuma has evolved by hybridization of species with different chromosome numbers of 2n = 24 and 18, resulting in a dibasic amphidiploid species.

Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita, important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India.

Root nodule bacterial strains were isolated from the little-studied legumes Eriosema chinense and Flemingia vestita (both in tribe Phaseoleae, Papilionoideae) growing in acidic soil of the sub-Himalayan region of the Indian state of Meghalaya (ME), and were identified as novel strains of Bradyrhizobium on the basis of their 16S rRNA sequences. Seven isolates selected on the basis of phenotypic characters and assessment of ARDRA and RAPD patterns were subjected to multilocus sequence analysis (MLSA) using four protein-coding housekeeping genes (glnII, recA, dnaK and gyrB). On the basis of 16S rRNA phylogeny as well as a concatenated MLSA five strains clustered in a single separate clade and two strains formed novel lineages within the genus Bradyrhizobium. The phylogenies of the symbiotic genes (nodA and nifH) were in agreement with the core gene phylogenies. It appears that genetically diverse Bradyrhizobium strains are the principal microsymbionts of these two important native legumes. The novel genotypes of Bradyrhizobium strains isolated in the present study efficiently nodulate the Phaseoloid crop species Glycine max, Vigna radiata and Vigna umbellata. These strains are genetically different from strains of Bradyrhizobium isolated earlier from a different agro-climatic region of India suggesting that the acidic nature of the soil, high precipitation and other local environmental conditions are responsible for the evolution of these newly-described Bradyrhizobium strains. In global terms, the sub-Himalayan region of India is geographically and climatically distinct and the Bradyrhizobium strains nodulating its legumes appear to be novel and potentially unique to the region.

Efficacy of RAPD, ISSR and DAMD markers in assessment of genetic variability and population structure of wild Musa acuminata colla.

North east India is considered as one of the major biodiversity hotspots worldwide and centre of origin of several plant species including Musa. Musa acuminata Colla is known to be one of the wild progenitors of cultivated bananas and plantains. Three single primer based DNA marker techniques viz., random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and directed amplification of minisatellites DNA (DAMD) were used for diversity diagnostics among 25 genotypes of wild M. acuminata collected from Meghalaya province of north east India. A total of 58 primers (26-RAPD, 21-ISSR, and11-DAMD) yielded 451 DNA fragments, of which 395 (87.58 %) were found to be polymorphic in nature. The polymorphic information content (PIC) values were almost identical for each marker system. The resolving power of the marker system was found to be highest in RAPD (3.96) whereas ISSR resolved highest marker index (16.39) in the study. Selected amplicon data obtained through single primer amplification reactions were utilized for determination of diversity within and among the populations of M. acuminata. Nei's genetic differentiation (Gst) value (0.451) indicated higher proportion of the genetic variation within the populations which is supported by the AMOVA analysis (88 %). The study provides insight into the efficacy of RAPD, ISSR and DAMD to analyse the genetic variation existing in the wild Musa germplasm, which can further be exploited for quality trait improvement and domestication of such important horticultural crops. The genetic diversity based population structure may shed light on the genetic basis of speciation and evolution of various species within the genus Musa.

Heterochromatin distribution and comparative karyo-morphological studies in Vignaumbellata Thunberg, 1969 and V.aconitifolia Jacquin, 1969 (Fabaceae) accessions.

Chromosome studies along with heterochromatin distribution pattern analysis have been carried out in two domesticated species of Vigna Savi, 1824 which grow in contrasting geo-climatic conditions of India: Vignaumbellata Thunberg, 1969, a legume well acclimatized to subtropical hilly regions of North-east India and Vignaaconitifolia Jacquin, 1969, a species of arid and semi-arid regions in desert plains of Western India. Karyo-morphological studies in both species reveal 2n = 22 chromosomes without any evidence of numerical variation and the overall karyotype symmetry in chromosome morphology suggest that the diversification at intraspecific level in genus Vigna has occurred through structural alteration of chromosomes, rather than numerical changes. Heterochromatin distribution as revealed by fluorochrome binding pattern using CMA3 and DAPI, confirms the occurrence of relatively more GC content in Vignaaconitifolia as compared to Vignaumbellata. However, AT content was found to be comparatively higher in Vignaumbellata which perhaps play a role in species interrelationships.

Genetic fidelity assessment in micropropagated plants using cytogenetical analysis and heterochromatin distribution: a case study with Nepenthes khasiana Hook f.

Rapid clonal propagation of selected genotypes has been one of the most extensively exploited approaches of biotechnology. However, inclusion of somaclonal variations in tissue-culture-derived plants results in the production of undesirable plant off-types which limits its applications in tissue culture industry. Therefore, the most critical concern has been the maintenance of genetic uniformity of micropropagated plants. Assessment of genetic fidelity in tissue-culture-raised plants of three consecutive regenerations of Nepenthes khasiana has been successfully carried out using chromosome counts and heterochromatin distribution pattern wherein changes in the number of chromosomes and the distribution of AT and GC base pairs were recorded. The cells studied in the plantlets of the first regeneration (23.33 %) showed deviant number of chromosome which was increased to 33.33 % and 40 % in the plantlets of the second and the third regenerations, respectively. Also, 4',6-diamidino-2-phenylindole (DAPI)(+) and chromomycin A3 (CMA)(+) binding sites, on an average of 5.74 ± 0.47 and 5.00 ± 0.30, were observed in the plantlets of the first regeneration. Subsequently, DAPI(+) binding sites were increased to 6.61 ± 0.39 and 6.74 ± 0.57 in the plantlets of the second and the third regenerations, respectively, with a corresponding decrease in the CMA(+) binding sites (4.63 ± 0.45 and 4.16 ± 0.47 CMA(+) sites in the plantlets of the second and the third regenerations, respectively). The study reveals an increase in cytological variations in the morphologically similar micropropagated plants of N. khasiana with the subsequent regenerations which further necessitate the determination of genetic integrity of micropropagated plants.

Comparative analysis of heterochromatin distribution in wild and cultivated Abelmoschus species based on fluorescent staining methods.

A comparative analysis of fluorochrome-binding pattern in nine taxa of Abelmoschus had shown that the type, amount and distribution pattern of heterochromatin were characteristic for each taxa. The fluorescent chromosome-binding sites obtained by chromomycin A3 (CMA) and 4',6-diamidino-2-phenylindole (DAPI) staining in all the nine species showed constitutive heterochromatin CMA(+), DAPI(+) and CMA(+)/DAPI(+). Large amount of heterozygosity was observed with regard to heterochromatin distribution pattern in all the taxa studied. The CMA(+)-binding sites are comparatively less than DAPI(+)-binding sites which is clearly evident as AT-rich regions are more than GC-rich regions in all the nine taxa analysed in Abelmoschus. These CMA(+) and DAPI(+)-binding sites apparently rise with the increased in chromosome numbers of the different species. This pattern of heterochromatin heterogeneity seems to be a general characteristic feature. Therefore, the differential pattern of distribution of GC- and AT-rich sequences might have played an important role in diversification of the genus Abelmoschus. Polyploidy is an important factor in the evolution of Abelmoschus and the sole reason for range in chromosome numbers in this genus. It may be noted that, though often, but not always, the increase of DNA is caused by an increase in the amount of heterochromatin, i.e. increase of non-coding sections indicating restructuring of the heterochromatin. Thus, cumulative small and direct numerical changes might have played a role in the speciation of Abelmoschus.

Heterochromatin characterization through differential fluorophore binding pattern in some species of Vigna Savi.

Heterochromatin regions are the most intensively studied and best known chromosome markers in plants. In Vigna species, blocks of constitutive heterochromatin were found either in the terminal or interstitial region of the chromosomes. The number and distribution of CMA(+) and DAPI(+) binding sites exhibit high chromosomal variability with characteristic unique banding patterns in all the eight taxa. A predominant feature was observed, i.e., most of the CMA(+) binding sites were in the terminal region of the short arm of some chromosomes while DAPI(+) binding sites were found mostly in the intercalary region of the chromosomes. The higher divergence in the heterochromatin blocks, as revealed by chromomycin A3 (CMA) binding pattern, in a few taxa, viz. Vigna glabrescens, Vigna khandalensis, and Vigna mungo, suggests that the processes of divergent evolution of repetitive sequences in genomic DNA involve a guanine-cytosine (GC)-rich region. On the contrary, Vigna dalzelliana had shown a prominent adenine-thymine (AT)-rich repetitive DNA sequence in terminal regions in the short arm of chromosomes while Vigna umbellata had shown in interstitial regions. The presence of prominent heterochromatic-rich regions, either GC- or AT-rich regions, does facilitate the rate of chromosomal rearrangements leading to restructuring of the karyotypes and thereby helping the species to attempt structural alterations as means of speciation.

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations.

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.

Genetic diversity and phylogenetic analysis of Citrus (L) from north-east India as revealed by meiosis, and molecular analysis of internal transcribed spacer region of rDNA.

The north-eastern region of India is reported to be the center of origin and rich in diversity of Citrus (L.) species, where some wild and endangered species namely Citrus indica, Citrus macroptera, Citrus latipes, Citrus ichagensis and Citrus assamensis exist in their natural and undisturbed habitat. In order to have comprehensive information about the extent of genetic variability and the occurrence of cryptic genomic hybridity between and within various Citrus species, a combined approach involving morphological, cytogenetical and molecular approaches were adopted in the present study. Cytogenetic approaches are known to resolve taxonomic riddles in a more efficient manner, by clearly delineating taxa at species and sub species levels. Male meiotic studies revealed a gametic chromosome number of n = 9, without any evidence of numerical variations. Bivalents outnumbered all other types of associations in pollen mother cells (PMCs) analyzed at diplotene, diakinesis and metaphase I. Univalents were frequently encountered in nine species presently studied, though their presence appropriately did not influence the distributional pattern of the chromosomes at anaphases I and II. The molecular approaches for phylogenetic analysis based on sequence data related to ITS 1, ITS 2 and ITS 1 + 5.8 s + ITS 2 of rDNA using maximum parsimony method and Bayesian inference have thrown light on species inter-relationship and evolution of Citrus species confirming our cytogenetical interpretations. The three true basic species i.e. Citrus medica, Citrus maxima and Citrus reticulata with their unique status have been resolved into distinct clades with molecular approaches as well. C. indica which occupies a unique position in the phylogenetic ladder of the genus Citrus has been resolved as a distinct clade and almost behaving as an out-group. The presences of quadrivalents in C. indica also echo and support its unique position. From our study it is amply clear that C. reticulata also has close relation to C. ichagensis, as these species have clustered together, denoting their close genetic relationship. On the other hand, our studies did not demonstrate a clear differentiation between subgenera Citrus and Papeda at the rDNA level. The combined approach of cytogenetical and molecular analysis did complement our early karyological findings and helped in resolving many a taxonomic riddles.

An invasive Mimosa in India does not adopt the symbionts of its native relatives.

BACKGROUND AND AIMS: The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species. METHODS: Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences. KEY RESULTS: Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the 'Old World' Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica. CONCLUSIONS: The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the 'local' Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S-5.8S-26S and 5S ribosomal RNA gene families, and the two tandemly repeated DNA sequences.

In the genus Carthamus (2n=20, 22, 24, 44, 64; x=10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S-26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S-26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n=20, and the two botanical varieties of B genome C. tinctorius (2n=24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S-26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n=20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n=44), and C. lanatus is one of the progenitors for the hexaploid (2n=64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2-10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.

Physical localization and probable transcriptional activity of 18S-5.8S-26S rRNA gene loci in some Asiatic Cymbidiums (Orchidaceae) from north-east India.

Fluorescence in situ hybridization based physical localization of 45S ribosomal DNA in eight horticulturally important species of Cymbidium (Orchidaceae) from north-east India (South-East Asia) has been carried for the first time. Observations revealed only one pair of chromosomes had NOR loci. Three, out of eight Cymbidiums showed decondensed, dispersed, extended form of hybridization signals of rDNA as dots of fluorescence (transcriptionally active), where as the rest of the Cymbidiums revealed condensed (non-active) forms, hence demonstrated the heteromorphism in size, intensities and their appurtenance which may be under epigenetic control. Except for the ribosomal genes, no other active genes have been reported to reside within the nucleoli. Such observations provide useful chromosome landmarks and provide valuable evidence about the genome evolution, speciation and ploidy both at molecular and chromosomal levels which is more or less highly ambiguous in family Orchidaceae.

Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of rDNA.

Sequence data obtained from nrITS region were used to assess phylogenetic inter-relationships and infrageneric classification of ten Cymbidium species collected from north-east India. The final aligned data matrix of combined ITS 1, 5.8S and ITS 2 yielded 684 characters. The ITS 1 and ITS 2 regions showed variable sequence lengths and G+C content (%). The 5.8S region was found to be more conserved (98.71%) followed by ITS 1 (86.12%) and ITS 2 (69.40%). ITS 2 recorded highest percentage of parsimony informative sites (7.46%), high sequence divergence with indels (24.63%), high number of transitions and transversions. ITS sequence data determined the phylogeny of Asiatic Cymbidiums with high bootstrap values. All three proposed subgenera could be distinguished clearly by all four (MP, ML, NJ, and BI) phylogenetic methods. This study validates the utility of ITS rDNA region as a reliable indicator of phylogenetic relationships, especially ITS 2 as probable DNA barcode at higher levels and can serve as an additional approach for identification of broader range of plant taxa especially orchids.

Single primer amplification reaction (SPAR) reveals inter- and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae).

A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456 polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observed polymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands which were either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to their entirely different habitats as well as discrete morphological characteristics. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPAR method revealed that analysis of natural genetic variation using combination of SPAR methods, rather than an isolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as the most powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD. Alternatively, the total polymorphic information content was highest in case of RAPD followed by other two SPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for genetic variation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservation concerns.

Karyological studies in ten species of Citrus(Linnaeus, 1753) (Rutaceae) of North-East India.

Ten Citrus (Linnaeus, 1753) species of North-East India have been karyo-morphologically analysed. All studied species had 2n=18 chromosomes without any evidence of numerical variation. All the chromosomes were found to be of metacentric and sub-metacentric in all the species; the morphology of the chromosomes showing size difference only. Symmetrical karyotype which does not have much difference in the ratio of longest to shortest chromosome in all the species was observed. Three species, Citrus grandis (Osbeck, 1757), Citrus reticulata (Blanco, 1837) and Citrus medica (Linnaeus, 1753) are identified as true basic species from asymmetry studies of karyotypes as they reflect on the primitive nature of their genomes. Citrus indica (Tanaka, 1937)occupies a special taxonomic position within the genus Citrus as a progenitor for other cultivated species.