Acta Pharmacol Sin 2008 Sep; 29 (9): 1127–1136 Full-length article Panax ginseng natural populations: their past, current state and perspec- tives1 Yuri N ZHURAVLEV2, Olga G KOREN, Galina D REUNOVA, Tamara I MUZAROK, Tatiyana Yu GORPENCHENKO, Irina L KATS, Yuliya A KHROLENKO Institute of Biology and Soil Science, Russian Academy of Sciences, 690022 Vladivostok, Russia Key words Abstract Panax ginseng; natural population; genetic Aim: The mating system of Panax ginseng, genetics and ontogenetic structure diversity; mating system; embryology; allozymes; AFLP; SSR of its natural populations of Primorye (Russia) were investigated. Methods: Genetic diversity was assessed using allozyme and the fluorescently based au- 1 This work was supported by grants from tomated amplified fragment length polymorphism (AFLP) and simple sequence the Russian Academy of Sciences (No 06-I- II11-028 and No 06-I-II11-030), of Russian repeats (SSR) markers. Results: Total genetic diversity at species level is low Foundation for Basic Research (No 06-04- with allozyme assay (0.023), and high with AFLP (0.255) and SSR (0.259) meth- 96054), and by the grant “Leading Schools of ods. It is observed within populations according to allozyme (>99%), AFLP Thought” of the President of Russian Federation (NSH6923-2006.4). (>85%), and SSR (>73%) assays. The indices of genetic variability distribution 2Correspondence to Yuri N ZHURAVLEV. point out the re-colonization of the Sikhote-Alin by ginseng plants from southern Phn 7-4232-31-0718 refuges during the warming period in the early Holocene. The capability of gin- Fax 7-4232-31-0193 seng plants to cross- and self-pollinate was shown and the assumption that Panax E-mail [email protected] ginseng is a facultative apomictic plant was confirmed. The reproductive system Received 2008-07-03 of ginseng possesses high plasticity and stability of the fertilization process that Accepted 2008-07-25 help the species to survive in stress conditions. Disturbances caused by external doi: 10.1111/j.1745-7254.2008.00866.x or internal factors can be reduced due to the morphogenetic potential of ginseng ovule or apomictic embryo development. Analysis of life stages structure of gin- seng 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 popu- lations studied are viable and the reintroduction of natural ginseng population is possible yet. [2] Introduction large forestlands of China, Korea and Russian Primorye , but nowadays its original distribution area has shrunken Panax ginseng C.A. Meyer is a well-known herb of to a few habitats in Russia and China. The largest, the Oriental traditional medicine, used by people for thousands Sikhote-Alin population, is located in the southern part of of years as a valuable remedy for various diseases and a the Sikhote-Alin mountain range. Another population is tonic drug for the elderly. located in the Nadezhdinsk and Khasan Districts of Primor- In ancient manuscripts (see review in Zhuravlev et al, [1] sky Kray, Russia (Khasan population), and Jilin and Hei- 1996 ) ginseng was already described as a rare plant. It longjiang Provinces, China, and a third, the Blue Mountain was introduced into culture in ancient China and Korea. population, is located in the Spassk District of Primorsky However, wild-growing ginseng was always considered to Kray, Russia. Any reports about findings of wild ginseng be of the most value. It was distributed in the Far Eastern in China have been rare. In Russia, ginseng was listed in forests on a wide territory, but because of intensive use of Red Book since 1975 as a threatened species[3]. Nowadays wild ginseng roots its area was drastically reduced, par- Primorsky Kray of Russia is the only place in the world ticularly in the last century. Indeed, in the first half of XX where natural ginseng populations are still alive and there- century wild-growing ginseng plants could be found on the fore our duty is to conserve this wonderful plant. Ginseng ©2008 CPS and SIMM 1127 Zhuravlev YN et al Acta Pharmacologica Sinica ISSN 1671-4083 populations are now close to extinction, and some urgent measures have been suggested to protect natural ginseng Material and methods resources from exhaustion[4]. Moreover, to change the Sampled populations Wild-growing Panax ginseng situation and to work out measures for protection of wild- plants were sampled from 3 non-protected natural popu- growing ginseng, in 1997 the Primorye regional admin- lations: Khasan (Kh), Blue Mountain (BM) and Sikhote- istration, Regional Committee of Natural Resources and Alin (SA). Collected living plants were transferred to an Institute of Biology and Soil Science of the Russian Acad- open experimental nursery in the natural ginseng habitats emy of Sciences (IBSS) elaborated the “Regional Complex (Spassky District of the Primorsky Kray) for further in- Long-term Program of Restoration (Reintroduction) of vestigations; a part of the plants was also transferred to the Primorye’s Ginseng Population up to 2005”. This program laboratory greenhouse. was based on many years’ study of various aspects of gin- Sample material Fresh leaves or those frozen in liquid seng biology conducted in IBSS. nitrogen were used for allozyme, amplified fragment length During the program execution, IBSS has been provid- polymorphism (AFLP), and simple sequence repeats (SSR) ing centers of reintroduction with scientific justification for analyses. A total of 206 plants of wild Panax ginseng were the selection and propagation of sampled material and for studied with allozyme analysis (34 from Kh, 49 from BM offspring identification, choice of number and emplacement and 123 from SA). For AFLP assay, 35 Panax ginseng of centers of reintroduction, search for more intact habi- plants were studied (10 from SA, 14 from BM, and 11 from tats for reintroduction and detailed recommendations for Kh), and 57 plants were studied with SSR markers (20 creation of reintroduced populations. During the program, from SA, 19 from BM, 18 from Kh). genetic analysis of extant ginseng populations, studies on For ontogenetic research, wild ginseng plants were pic- their morphological features and mating system were car- tured and their above-ground organs were collected, dried ried out to understand the processes taking place in ginseng and studied. populations. Mating system experiments were conducted in the Ginseng is known to be a relic of the Tertiary era[2] and laboratory greenhouse and the nursery over 4 years. For its prosperity in extant habitats depends on its individual pollination experiments and embryology studies, flowering ability to adapt to the environment. Estimation of genetic 5–25-year-old ginseng plants from natural populations and and morphological variability, heterogeneity of popula- 5-year-old cultivated plants were used. tions, and differentiation within and among populations Allozyme analysis Enzyme extraction, electrophore- are usually the best way to describe the state of an endan- [5,6] sis, enzyme staining and interpretation were carried out as gered species . At the same time, genetic variation of [10] described in Koren et al . Enzymes were extracted from plant populations is known to be largely determined by fresh leaves or those frozen in liquid nitrogen. evolutionary history and mating system of the species[7]. AFLP and SSR analysis Genomic DNA was extracted Therefore, elucidation of these items would be useful for [11] according to Echt et al and then purified by Murray and understanding both the current state and perspectives of [12] Thompson . natural ginseng populations. Moreover detailed study of Amplified fragment length polymorphism analysis was the ginseng mating system and embryology is urgent for [13] conducted according to Vos et al . DNA was digested us- propagation of sampling ginseng plants in reintroduction ing the restriction endonucleases EcoRI and MseI. AFLP centers. Significant items of the conservation program are primers for pre-amplification contained 2 selective nucle- an evaluation of the current state of ginseng populations, otides. The EcoRI primers contained 3 selective nucle- including numbers, age composition and viability, and otids and 4 selective nucleotids were used for the MseI analysis of spatial and temporal variability of coenopopu- primers. AFLP adapters and primers were synthesized by lations. Nowadays ontogenetic analysis of populations is “SibEnzim” (Russia). Selectively amplified products were widely used for resource estimation of medicine plants[8,9]. analyzed using the ABI 3100 automated DNA sequencing Such investigations should be conducted to forecast the system (Applied Biosystems, USA). Each of the EcoRI- fate of the exhausted natural populations of Panax ginseng. NNN primer types was labeled with FAM fluorescent dye In this paper, we summarize the results of our investi- at the 3' end. Size alignment of the AFLP fragments was gations on the ginseng mating system, genetics and onto- carried out with ABI GeneScan Analysis Software (Applied genetic structure of its natural populations for evaluation of Biosystems) with the ABI GeneScan 500 LIZ internal lane its current state and perspectives. 1128 Http://www.chinaphar.com Zhuravlev YN et al size standard. DNA fragments ranging from 50 to 500 bp velopment we used the clearing technique for angiosperm in size from the AFLP analysis were scored. ovules[21]. Examination of autofluorescence in ovules was Primers designed to flank 2 microsatellite loci (CT12, carried out with a LSM 510 META confocal laser scanning CA33) isolated from the ginseng genome were 5'-GAGAG microscope (Carl Zeiss, Germany) equipped with argon TAACC ACAGG ATAGA GAAA-3' (CT12 F), 5'-CTCCC laser. Confocal z-stack images and single plans were ob- TTTAC AGGTA GATAG TGAA-3' (CT12 R), and 5'- tained after excitation at 488 nm and emission at 522 nm CGATG TGGAT TTCAA TTTTA AG-3' (CA33 F), 5'- using a 505-nm LP filter.