In-vitro Propagation, Conservation And ISSR Analysis In Some Species And Types Of Rhododendron In Fujian

In this experiment Rhododendron was used as the materials for the following5aspects of researches:①using the flower buds, leaves and stem sections of Rhododendron pulchrum and Rhododendron simiss asexplants, plant regeneration systems were established;②collection of Rhododendron germplasm resourcesin Fujian such as Rhododendron pulchrum, Rhododendron simiss, etc. were conducted;③the effects ofdifferent concentrations of sucrose, agar, mannitol, ABA, PP333and pH values on in vitro conservation ofplantlets were compared by single factor design;④determination of SOD and POD activities during invitro conservation was carried out in Rhododendron spp. cv. Erqiaodujuan;⑤the cluster analysis of ISSRmarkers of Rhododendron species from Jiuxian Mountain in DaiYun mountains, JiuFeng Mountains, theSoutheast Coastal and the genetic diversity analysis of ISSR markers of Rhododendron latoucheae andRhododendron simiss from different altitudes of Jiuxian Mountain were conducted. The main results were asfollows:1. In vitro Propagation of some species of Rhododendron germplasm resources in Fujianprovince.①Development of disinfection method and establishment of the aseptic plantlet system inRhododendron pulchrum.The flower buds were better than the leaves and stems as the explants for establishment of theregeneration system, with the induction rate as high as93.33%in Rhododendron pulchrum. With the samedisinfection method, the disinfection effects were different, and the contamination rate of stem sections washigher than that of the leaves, but the rate of browning or mortality was lower. There was no difference in thesurvival rates. The disinfecting processing of flower buds was:75%alcohol (30s)+0.1%HgCl2(12min)+sterile-water5times; the disinfecting processing of leaves was:75%alcohol (30s)+10%NaClO(3min)+sterile-water3times+0.1%HgCl2(6min)+sterile-water5times; the disinfecting processing of stem sectionswas:75%alcohol disinfection (30s)+10%NaClO(6min)+sterile-water3times+0.1%HgCl2(8min)+sterile-water5times. After disinfection processing, the explants were inoculated on the solid medium—WPM+1.0mg/L ZT+30g/L sucrose+6g/L agar(pH5.4)for induction culture. The establishment of theaseptic plantlet system was similar in Rhododendron simiss. ②Proliferation culture of Rhododendron pulchrum and Rhododendron simsii.Through the DPS software and with3-level and4-factor orthogonal design, the effects of the basalmedium, concentrations of ZT, NAAand sucrose on the proliferation were compared in Rhododendron simsiiand Rhododendron pulchrum, respectively. The results showed that: the lower salt medium such as1/2WPMmedium was suitable for proliferation in Rhododendron; ZT and NAA was sensitive for proliferation andhigher concentrations of ZT (﹥2mg/L) were beneficial to the Rhododendron induction and proliferation;the effect of high levels of NAA were adverse. Comprehensive consideration with the test result, the idealculture medium formula was1/2WPM+4.0mg/L ZT+20g/L sucrose+6g/L agar for Rhododendron simsii.,and1/2WPM+2.0mg/L ZT+0.05mg/L NAA+20g/L sucrose+6g/L agar for Rhododendron pulchrum.③Rooting culture of Rhododendron pulchrum and Rhododendron simsii.Test in Rhododendron rooting culture showed that NAA, IBA concentrations within a certain range werebeneficial to rooting in Rhododendron; NAA was more sensitive than IBA, and the high concentration of NAAimproved rooting obviously. The ideal rooting medium was1/2WPM+1.5mg/L NAA+20g/L sucrose+6g/L agar for Rhododendron simsii, and1/4WPM+0.5mg/L NAA+1.5mg/L IBA+30g/L sucrose+6g/Lagar for Rhododendron pulchrum. Therefore, the suitable rooting medium for different Rhododendron specieswas slightly different.④Optimization of rooting conditions in Rhododendron spp. cv. Erqiaodujuan.Using the tube plantlets as the experimental materials, the optimization of rooting conditions was carriedout. The result: showed that the best rooting medium was1/4WPM+1.5mg/L NAA+20g/L sucrose+6g/Lagar. After transplant into turfy soil, the plantlets grew normally.2. In vitro conservation of plantlets and the analyses of the changes of physiological andbiochemical indexes during in vitro conservation in Rhododendron spp. cv.Erqiaodujuan.①The effects of different concentrations of sucrose, agar, mannitol, ABA, PP333and pH values on in vitroconservation for200d were analyzed by single factor design in Rhododendron spp. cv. Erqiaodujuan. Theresult showed that the best conservation medium was the medium supplemented with30g/L sucrose,7.5g/Lagar,0.5mg/L ABA,20mg/L PP333and with the pH value of6.5, and the survival rate was up to75%afterconservation for200d.②The determination of SOD and POD activities during in vitro conservation showed the curve rose at theearly stage, and then dropped after maintenance for some time.3. ISSR analysis of some species of Rhododendron germplasm resources in Fujian area.The genetic diversity of13accessions of Rhododendro germplasm resources in Fujian from different areas and different species was assessed by ISSR, and the results showed: the genetic similarity coefficient(GS) of13Rhododendron accessions in Fujian between0.3235and0.7647, the range was relatively large.Average GS was0.5294, the genetic distance was0.4706, which showed that the genetic difference was verysignificant in different areas of Fujian Province, and there existed high degree of differentiation amongspecies.Using NTSYS software the dendrogram of13Rhododendron accessions in Fujian was built. At geneticsimilarity coefficient of0.498, they could be divided into three groups: the first-group included Rhododendronlatoucheae in Jiuxian Mountain, No.2of Rhododendron simsii in Gushan mountain; the second-group includedRhododendron simsii in Jiuxian Mountain, No.1of Rhododendron simsii in Gutian, No.2of Rhododendronsimsii in Gutian, No.3of Rhododendron simsii in Gutian, No.4of Rhododendron simsii in Gutian, No.1ofRhododendron hybridum, No.2of Rhododendron hybridum No.1of Rhododendron simsii in Gushan mountain;the third-group included Rhododendron ovatum, Rhododendron pulchrum, Rhododendron spp. Erqiaodujuan.At0.57, they could be divided into5groups: The first-group included Rhododendron latoucheae in JiuxianMountain, No.2of Rhododendron simsii in Gushan Mountain; the second-group included Rhododendron simsiiin Jiuxian Mountain, No.1of Rhododendron simsii in Gutian, No.2of Rhododendron simsii in Gutian, No.3ofRhododendron simsii in Gutian, No.2of Rhododendron hybridum, No.1of Rhododendron simsii in Gushanmountain; The third-group included No.4of Rhododendron simsii in Gutian, No.2of Rhododendron hybridumThe fourth-group includeds Rhododendron ovatum, the fifth-group included Rhododendron pulchrum andRhododendron spp. Erqiaodujuan.The analyses of ISSR molecular marker indicated that the genetic similarity coefficient (GS) of the28accessions of Rhododendron latoucheae and Rhododendron simsii from different elevations in Jiuxian Mountain,was between0.457and0.920, which suggested that there exist greater genetic difference among different plantsfrom different altitudes in Jiuxian Mountain in the two Rhododendron species, which might result from theenvironmental or climatic conditions at the different altitudes. At genetic similarity coefficient of X1=0.55, the28accessions of Rhododendron could be divided into two groups: the first-group included the lower altitude ofRhododendron latoucheae in Jiuxian Mountain,the middle altitude of Rhododendron latoucheae in JiuxianMountain,the higher altitude of Rhododendron latoucheae in Jiuxian Mountain；the second-group included thelower altitude of Rhododendron simsii in Jiuxian Mountain, the middle altitude of Rhododendron simsii in JiuxianMountain, the higher altitude of Rhododendron simsii in Jiuxian Mountain; at genetic similarity coefficient ofX1=0.61, the28accessions of Rhododendron could be divided into two sub-groups: the first-group included thelower altitude of Rhododendron simsii in Jiuxian Mountain,the higher altitude of Rhododendron simsii in JiuxianMountain；the second-group included the middle altitude of Rhododendron simsii in Jiuxian Mountain；the third-group included the lower altitude of Rhododendron latoucheae in Jiuxian Mountain；the middle altitude ofRhododendron latoucheae in Jiuxian Mountain；the fourth-group included the higher altitude of Rhododendronlatoucheae in Jiuxian Mountain；at genetic similarity coefficient of X1=0.63, the28accessions of Rhododendroncould be divided into six groups: the lower altitude of Rhododendron latoucheae in Jiuxian Mountain,themiddle altitude of Rhododendron latoucheae in Jiuxian Mountain,the higher altitude of Rhododendronlatoucheae in Jiuxian Mountain, the lower altitude of Rhododendron simsii in Jiuxian Mountain,the middlealtitude of Rhododendron simsii in Jiuxian Mountain, the higher altitude of Rhododendron simsii in JiuxianMountain.