T. I. Muzarok

Isoflavonoid production by callus cultures of Maackia amurensis

Callus cultures were established from the different parts of Maackia amurensis plants and analyzed for isoflavonoids. The isoflavones daidzein, retuzin, genistein and formononetin and the pterocarpans maakiain and medicarpin were found to be produced by these cultures. The content of isoflavones and pterocarpans was essentially the same in cultures derived from leaf petioles, inflorescences and apical meristems of the plant. The maximal yield of isoflavones and pterocarpans in calluses was 20.8 mg/g cell dry wt., approximately four times higher than the content of the heartwood of M. amurensis plants. Unlike wild-growing plants, none of the cell cultures had the ability to accumulate stilbenes.

Polymorphism of RAPD, ISSR and AFLP markers of the Panax ginseng C. A. Meyer (Araliaceae) genome

The genus Panax (Araliaceae) is world-famous because many its members have important medicinal properties. Panax ginseng C. A. Meyer is more popular than other species of the genus because remedies prepared from this plant stimulate immunity, help to prevent diseases, and have antistress effects. In addition, the ginseng root extract is traditionally used as a means against aging. At present, this species is found in the wild only in Primorsky krai, Russia, but its populations are extremely exhausted and need to be restored. In this study, effectiveness of molecular DNA markers in detecting genetic variation and differentiation of the ginseng populations was tested. Genetic variation of ginseng, identified using RAPD (P = 4%; Hpop = 0.0130) and ISSR (P = 9.3%; Hpop = 0.0139) markers was low. The AFLP* approach, according to which amplicons are separated in polyacrylamide gel and visualized by means of silver staining, showed somewhat higher variability (P = 21.8%; Hpop = 0.0509), while its effectiveness in population differentiation was as low as that of RAPD and ISSR. The AFLP** technique, which included analysis of the fragments using genetic analyzer, revealed high genetic diversity of ginseng (P = 94.4%; Hpop = 0.3246). All populations examined using the AFLP** markers were statistically significantly differentiated based on the AMOVA results. Our result suggest effectiveness of AFLP** markers for characterization of the genetic structure and genetic relationships of the ginseng populations. These markers are recommended for use in large-scale population genetic studies of this species to develop measures of its conservation.

RAPD and Allozyme Analysis of Genetic Diversity in Panax ginseng C.A. Meyer and P. quinquefolius L.

Inter- and intraspecific variation of two ginseng species Panax ginseng and P. quinquefolius was estimated by studying 159 RAPD and 39 allozyme loci. Parameters of polymorphism and genetic diversity were determined and a tree was constructed to characterize the differences between individual plants, samples, and species. Genetic variation in P. ginseng proved to be lower than in P. quinquefolius. Gene diversity in the total P. ginseng sample was comparable with the mean expected heterozygosity of herbaceous plants. This suggests that wild P. ginseng plants in various areas of the currently fragmented natural habitat and cultivated plants of different origin have retained a significant proportion of their gene pool. The mean heterozygosity calculated per polymorphic locus for the RAPD phenotypes is similar to that of the allozyme loci and may be helpful in estimating gene diversity in populations of rare and endangered plant species.

RAPD Analysis of Genome Variability of Planted Ginseng, Panax ginseng

Genome variability of 23 ginseng plants (Panax ginseng) grown in culture in Primorskii Krai was studied by RAPD method. Eleven arbitrary chosen primers were used to analyze 138 loci of DNA samples, 17 of which appeared to be polymorphic. The OPD-11-1000 fragment was found to be a RAPD marker allowing plants to be differentiated according to their morphotype. Using five primers, it was demonstrated that the genetic polymorphism of the cultivated plants is lower than that in nature (7.6% and 10.6%, respectively). Dendrograms of genetic relatedness are in accord with genetic differences between individuals of plantedP. ginseng belonging to different morphotypes, and demonstrate close relatedness of one of the morphotypes to wild plants. This morphotype could be recommended for reintroduction into natural habitats.

Age stages in the ontogeny of cultivated Panax ginseng C.A. Mey

The ontogeny of perennial polycarpic herb Panax ginseng C.A. Mey. (Araliaceae) under plantation conditions was described. Three periods (latent, pregenerative, and generative) and eight age stages have been identified in the ontogeny of cultivated P. ginseng. The generative period of this species is the longest ontogenetic period, which determines the timing of its cultivation in plantations.

Population genetic structure of wild-growing ginseng ( Planax ginseng C.A. Meyer) assessed using AFLP markers

Genetic variability in ten populations of wild-growing ginseng was assessed using AFLP markers with the application of fragment analysis on a genetic analyzer. The variation indices were high in the populations (P = 55.68%, HS = 0.1891) and for the species (P = 99.65%; HS = 0.2857). Considerable and statistically significant population differentiation was demonstrated (θB = 0.363; Bayesian approach, “full model”; FST = 0.36, AMOVA). The results of AMOVA and Bayesian analysis indicate that 64.46% of variability is found within the populations. Mantel test showed no correlation between the genetic and geographic distances among the populations (r = −0.174; p = 0.817). Hierarchical AMOVA and analysis of genetic relationships based on Euclidean distances (NJ, PCoA, and MST) identified two divergent population groups of ginseng. Low gene flow between these groups (Nm = 0.4) suggests their demographic independence. In accordance to the concept of evolutionary significant units (ESU), these population groups, in terms of the strategy and tactics for conservation and management of natural resources, should be treated as management units (MUs). The MS tree topology suggests recolonization of southern Sikhote-Alin by ginseng along two directions, from south and west.

Pollen Heteromorphism in Panax ginseng C.A. Meyer (Araliaceae) Anthers

76 Like in animals, the development of plant male gametes is a lasting process of a gradual structural conversion of early gametes into late ones [1, 2]. The stages of P. ginseng pollen grain development from archesporium to the stages of microspore germination, generative cell division, and sperm formation were described using light microscopy [3, 4]. These studies established that mature ginseng pollen grains have exine with three pores and contain a vegetative and two generative cells. In research using light and scanning and transmission electron microscopy, exine morphology and ultrastructure were examined for pollen of P. ginseng and other species of the genus Panax [5] . Morphological characteristics of pollen grains and ultrastructure of exine are traditionally used in taxonomic and phylogenetic plant studies [5–8]. Wen and Nowicke [5] described and illustrated exine ultrastructure for ten Panax species and concluded that the palynological analysis does not confirm the monophyly of the group Panax species including P. ginseng, P. notoginseng , and P. quinquefolius , although close relationship between them was stated on the basis of plant morphology and the triterpenoid qualitative analysis [9]. Similarly, close sister-pair relationship between P. ginseng and P. quinquefolius suggested by Li [10] was not supported.