Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

BACKGROUND: Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. METHODS: The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. RESULTS: In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. CONCLUSION: This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Characterization of the Variability of Nucleoli in the Cells of Panax ginseng Meyer In Vivo and In Vitro.

Results of karyological study of intact plants and some callus lines of Panax ginseng are presented. In the native plants of P. ginseng the nucleus with 1 nucleolus (90%) dominate, and nucleus with 2 nucleoli is rare. One nucleolar nucleus also dominate in interphase nuclei of cells of cultivated P. ginseng (from 2006), but we also found nucleus with 2 to 3 nucleoli in the same cell lines. Interphase nuclei of P. ginseng in long cultivated lines (from 1988) contain 1 to 9 nucleoli, with a predominance of nuclei containing from 3 to 4 nucleoli. It was shown that long-time cells (cultivated since 1988) had cytogenetic changes such as increase level of polyploid and aneuploid cells, increase of nucleoli number into interphase nucleus and decrease of nuclei/nucleoli ratio. These long-time cultivated cells had very low ginsenoside content.

Genetic variability and population structure of endangered Panax ginseng in the Russian Primorye.

BACKGROUND: The natural habitat of wild P. ginseng is currently found only in the Russian Primorye and the populations are extremely exhausted and require restoration. Analysis of the genetic diversity and population structure of an endangered species is a prerequisite for conservation. The present study aims to investigate the patterns and levels of genetic polymorphism and population structures of wild P. ginseng with the AFLP method to (1) estimate the level of genetic diversity in the P. ginseng populations in the Russian Primorsky Krai, (2) calculate the distribution of variability within a population and among populations and (3) examine the genetic relationship between the populations. METHODS: Genetic variability and population structure of ten P. ginseng populations were investigated with Amplified Fragment Length Polymorphism (AFLP) markers. The genetic relationships among P. ginseng plants and populations were delineated. RESULTS: The mean genetic variability within populations was high. The mean level of polymorphisms was 55.68% at the population level and 99.65% at the species level. The Shannon's index ranged between 0.1602 and 0.3222 with an average of 0.2626 at the population level and 0.3967 at the species level. The analysis of molecular variances (AMOVA) showed a significant population structure in P. ginseng. The partition of genetic diversity with AMOVA suggested that the majority of the genetic variation (64.5%) was within populations of P. ginseng. The inter-population variability was approximately 36% of the total variability. The genetic relationships among P. ginseng plants and populations were reconstructed by Minimum Spanning tree (MS-tree) on the basis of Euclidean distances with ARLEQUIN and NTSYS, respectively. The MS-trees suggest that the southern Uss, Part and Nad populations may have promoted P. ginseng distribution throughout the Russian Primorye. CONCLUSION: The P. ginseng populations in the Russian Primorye are significant in genetic diversity. The high variability demonstrates that the current genetic resources of P. ginseng populations have not been exposed to depletion.

The pollen metamorphosis phenomenon in Panax ginseng, Aralia elata and Oplopanax elatus; an addition to discussion concerning the Panax affinity in Araliaceae.

To find more morphological characteristics useful for discussion on aralian or non-aralian Panax affinity, pollen morphological diversity was comparatively analysed in P. ginseng, Aralia elata and Oplopanax elatus collected during their pollination periods. In the anthers of both the buds and open flowers, the pollen average diameter varied between some species-specific maximum and minimal measurement. However, the larger pollen grains were typically found in the buds whereas the smaller pollen prevailed in the open flowers, testifying to the pollen size diminution during anther maturation. Based on this finding, the subsequent examination of pollen according to size decrease was put into operation as a method of pollen modification for the study. The structural mechanisms of pollen metamorphosis were identified as not being species specific but rather universal. These mechanisms are suggested to be the shrinkage of the pollen vegetative cytoplasm, the intine enlargement, the deepening of three colporate apertures provided by exine sunken into enlarged intine areas, the aperture accretion as well as the transformation of the exine from thick/sculptured into thin/less sculptured. During 'size-reducing metamorphosis', the pollen grains changed dramatically, going through a species-specific set of intermediate morphs to the final species-specific morphotype. In P. ginseng this morphotype is round (diameter is about 16 microm), in A. elata it is round with a single projection (diameter is about 15 microm) and in O. elatus it is ovoid with a single projection (average diameter is about 18 microm). In addition, every species is peculiar in having the unique vegetative cytoplasm inclusions and individual construction of the largest pollen exine. From a phylogenetic perspective, these findings presumably add support to the option of equal remoteness of P. ginseng from A. elata and O. elatus. The characteristics found seem to be suitable for examination of Panax affinity, by the subsequent study of more Araliaceae representatives.

Panax ginseng natural populations: their past, current state and perspectives.

AIM: The mating system of Panax ginseng, genetics and ontogenetic structure of its natural populations of Primorye (Russia) were investigated. METHODS: Genetic diversity was assessed using allozyme and the fluorescently based automated amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers. RESULTS: Total genetic diversity at species level is low with allozyme assay (0.023), and high with AFLP (0.255) and SSR (0.259) methods. It is observed within populations according to allozyme (> 99%), AFLP (> 85%), and SSR (> 73%) assays. The indices of genetic variability distribution point out the re-colonization of the Sikhote-Alin by ginseng plants from southern refuges during the warming period in the early Holocene. The capability of ginseng plants to cross- and self-pollinate was shown and the assumption that Panax ginseng is a facultative apomictic plant was confirmed. The reproductive system of ginseng possesses high plasticity and stability of the fertilization process that help the species to survive in stress conditions. Disturbances caused by external or internal factors can be reduced due to the morphogenetic potential of ginseng ovule or apomictic embryo development. Analysis of life stages structure of ginseng populations demonstrates that all of them are not full-constituents because some life stages are absent or occur rarely. CONCLUSION: In all 3 populations, virgin and young generative individuals are predominant. This means that populations studied are viable and the reintroduction of natural ginseng population is possible yet.