Fluorescence In Situ Hybridization And Karyotype Analysis Of Three Medicinal Plants Of Panax Species

P.ginseng,P.quinquefolius and P.notoginseng,three representative medicinal plants of the P.species,had attacted scholars' great cosideration for their high medicinal and economic value.However,current studies on these medicinal plants concertrated more on the aspects of cultivation,physiology,pharmacology and chemical pharmacy than on their genetic characteristics,which is the reason that the application of some molecular biotechnology in the medicinal plants of the P.species has been restricted.This paper made further optimization towards the chromosome preparation technology with different meristems of these plants as materials,and analyzed their karyotype and phylogenetic relationship through double FISH method with rDNA sequences screened by PCR method as probes.Following are the main results.1.Looking for the best condition of chromosome preparation for each material through comparing different factors influencing on this technology.The results showed that sampling was the key of chromosome preparation.With roots induced by tissue culturing plantlets growing to 2cm,roots of seeds growing to 1.5cm,roots induced by water culturing ginseng plantlets growing to 1cm and callus subcultured for 6-9d,the metaphase cell amount in these materials had been demonstrated to be the most suitable.As for chromosome form,it was better when the roots of all three kinds were pretreated 2-6h in 2mmol/L 8-hydroxyquinoline or 10-15 h in 0? ice water while callus placed in 0? for 24 h..As for enzymolysis consequence,the chromosome would be much separated with a clean background when the roots of tissue culturing plantlets and the roots of ginseng were hydrolyzed by 2% cellulase and 1%pectinase for 90 min in 37?.while the roots of seeds and callus were hydrolyzed for 120 min.It could get some good metaphase chromosome that chromosome squashing was stained with Carbol fuchsin solution or 45% acetic acid.Drop-spreading method was suitable for preperation of seed roots while squashing method was suitable for preperation of other materials.2.Practicing and perfecting the use of FISH on medicinal plants of P.species with rDNA probes screened by PCR method.Among the 8 rDNA probes obtained,p18 S,p45S1 and p45S2,which were highly conserved,each had a pair of hybrid sites on the chromosomes of all three species.p5S1 probe had two pairs of hybrid sites each on the chromosomes of P.ginseng and P.quinquefolius while p5S2 probe had only one.p5S3 and p5S5 each had two pairs of hybrid sites on the chromosomes of P.ginseng and P.quinquefolius and one pair on the chromosomes of P.notoginseng.p5S4 had one pair on the chromosomes of P.quinquefolius.3.Analyzing the karyotypes of P.ginseng,P.quinquefolius and P.notoginseng along with the relationships between them by using double FISH with probes which had better hybridization signals and sufficient hybridization sities and refered to the characteristic parameters of chromosomes and rDNA distribution sites.Here are the results in detail.The karyotype formula of P.ginseng was2n=48=30m+14sm+2st+2T,with a pair of clear hybridization sites of p18 S on the satellites of its 1st chromosomes and two pairs of hybrid signals of p5S1,one pair of which were strong and located in the short arms near the centromere position of 14 th chromosomes while the other one pair were weak andlocated in the long arms next to the centromere position of 3rd chromosomes.The karyotype formula of P.quinquefolius was 2n=48=22m+22sm+2st+2T,with a pair of clear hybridization sites of p18 S on the short arms of 5th chromosomes and two pairs of hybrid signals of p5S3,one pair of which were strong and located in the short arms near the centromere position of 9th chromosomes while the other one pair were weak and located in the long arms next to the centromere position of 6th chromosomes.The karyotype formula of P.notoginseng was 2n=24=20m+4sm,with a pair of clear hybridization sites of p18 S on the middle of short arms of 1st chromosomes and a pair of hybridization sites of p5S3 on the middle of short arms of 11 th chromosomes.From cluster analysis of karyotype similarity and the characteristics of rDNA distribution sites,a conclution had been drawn.That was,a close relationship had been found between P.ginseng and P.quinquefolius while P.notoginseng was far from them.