Development And Optimization For Rapid Propagation In Vitro Of Flowering Season Rhododendron Ancient Trees From Pingnan

There are two ancient azaleas trees which can bloom during four seasons in Pingnan county of Fujian Province,one belongs to Rhododendron pulchrum Sweet and is more than 400 years located in Longyuan Village of Kangkou Town,another belongs to Rhododendron simsii Planch and is about 560 years located in Jiufeng Temple of Xiling Town.They are facing extinction because of accumulating more endogenous bacteria or fungi,and attaching surface parasites under conditions of poor growth and nutrition environments and have high physiological age and weak meristematic ability.Conventional and open rapid propagation in vitro of the ancient trees were developed and optimize with terminal buds as explants taken from outside shoots of the crowns,the optimal sampling seasons and surface disinfection conditions of explants were determined,the optimal combinations of microbial inhibitors in open rapid propagation in vitro,the methods of rescuing of contaminated explants,the optimal medium components in different stages of rapid propagation in vitro including induction of adventitious buds,subculture multiplication of cluster buds,culture of strengthening shoots,rooting culture of rootless shoots,and optimal medium ratio for transplating for rooted seedlings,which will provide supports for protection,exploitation,and industrial production of four-season ancient rhododendron trees,and have important practical values.The results were as follows:1.Conventional rapid propagation system in vitro was developed and optimized in Rhododendron pulchrum SweetTerminal buds were taken as explants from shoots in different growth seasons for screening the best disinfection,the results showed that the optimal sampling seasons for 430a ancient tree and five years tree of Rhododendron pulchrum Sweet were mid-May and mid-April,respectively,their optimal sterilizing time of both explants was 8minutes,which could result in 44.6%and 50%at success rate.Contaminated explants for 430a ancient tree in sterile culture were rescued,the results indicated that the treated explants were dipped into75%ethanol solution for 20 seconds,then were transferred into 0.1%mercury bichloride solution for 5 minutes,and were transferred into fresh basic medium for 4 times and the success rate reached 71%.The resulted sterile explants inducing adventitious buds,the results showed that the optimal combination of induction media for 430a ancient tree and five years tree of Rhododendron pulchrum Sweet were WPM medium addition of 3.0 mg/L of zeatin?ZT?and 0.1 mg/L of naphthylicetic acid?NAA?and 1/4MS medium addition of 3.0 mg/L of ZT and 0.1 mg/L of NAA,which resulted in 3.45 and 2.85 in number of valid buds,respectively.The top buds and stem segments with axillary buds from resulted sterile shoots were induction of cluster buds,the results showed that the stem segments with axillary buds were more suitable than top buds for induction of multiplication of cluster buds in 430a ancient tree and five years tree of Rhododendron pulchrum Sweet,and the optimal basic medium were Anderson and WPM respectively,the cytokinin were TDZ,and the optimal subculture multiplication medium of 430a ancient tree was Anderson addition 0.5mg/L of TDZ and 0.1 mg/L of NAA whose multiplication coefficient was 13.23 which was 1.4 times of that of top buds,those of five years tree was WPM addition 1.0 mg/L of TDZ and0.1 mg/L of NAA whose multiplication coefficient was 9.67 which was2.75 times of that of top buds.The clustered shoots were strengthen seeding culture,the results showed that the optimal strengthening culture medium was WPM addition 0.5 mg/L of ZT and 0.5 mg/L of GA₃ whose culture cycle was about 60 days for ancient tree;that for five years tree was WPM addition0.5 mg/L of ZT,0.5 mg/L of NAA,and 0.1mg/L of GA₃ whose culture cycle was about 70 days.The rootless shoots were inoculated on rooting media,the results indicated that the optimal rooting medium for both trees was WPM addition 0.1 mg/L of ZT and 0.5 mg/L of NAA,the rooting rate for ancient tree was 92.6%,that for five years tree was 98.6%.2.Conventional rapid propagation system in vitro was developed and optimized in Jiufeng Temple 560a ancient tree of Rhododendron simsii PlanchTerminal buds were taken as explants from shoots in different growth seasons,after sterilize the results showed that the optimal sampling seasons for Rhododendron simsii Planch ancient tree were mid-May,respectively,which success rate attached 60.39%.The contaminated explants for Rhododendron simsii Planch ancient tree were disinfection again,the results indicated that the treated explants were dipped into 75%ethanol solution for 20 seconds,then were transferred into 0.1%mercury bichloride solution for 5 minutes,and were transferred into fresh basic medium for 4 times,which will result in 65%at success rate for rescue of contaminated explants.The sterile explants inducing adventitious buds,the results showed that the optimal combination of induction media for ancient tree of Rhododendron simsii Planch were WPM medium addition of 3.0 mg/L of ZT and 0.1 mg/L of NAA,which resulted in 3.20 in number of effective buds.The top buds and stem segments with axillary buds of Rhododendron simsii Planch ancient treewere induction cluster buds,the results showed that the stem segments with axillary buds were more suitable than top buds for induction of multiplication of cluster buds;that the optimal subculture multiplication medium was WPM addition 3.0mg/L of ZT and 0.1 mg/L of NAA and 0.5 mg/L GA₃,whose multiplication coefficient was 3.35,which was 2.2 times of that of top buds.The resulted clustered shoots of Rhododendron simsii Planch ancient tree for strong seeding,the results showed that the optimal strengthening culture medium was WPM addition 0.05 mg/L of ZT and 0.05 mg/L of GA₃ whose culture cycle was about 60 days.3.Open rapid propagation system in vitro was developed and optimized in Rhododendron pulchrum SweetThe WPM basic medium was exposed to open natural environment or was innoculated with fungi and bacteria for screening kinds and valid concentration of microbial inhibitors.The results showed that optimal concentration of microbial inhibitors under condition of natural exposure were 0.005%of sodium hypochlorite and 100 mg/L of mancozeb;that under condition of artificial inoculation of microbes?bacteria and fungi?,the valid concentrations of microbial inhibitors for bacteria were 0.01%of sodium hypochlorite and 100 mg/L mancozeb,those for fungi were0.15%of sodium hypochlorite and 0.20%sodium hypochlorite and more than 600 mg/L of mancozeb.The explants sterilized were inoculated on media for open culture,the results showed that the optimal method for development of sterile cultures in open culture was that explants sterilized for 15 minutes with0.1%of sodium hypochlorite were innoculated on media for open culture addition of 0.1%of sodium hypochlorite as microbial inhibitors,whose success rate were 60%and 46.67%in five years and 430a ancient tree of Rhododendron pulchrum Sweet which were higher that those for conventional culture in vitro.The contaminated explants in development of sterile cultures for open culture in vitro were resterilized for open culture,the results indicated that the contaminated explants which were fistly sterilized for30 seconds with 75%ethanol and were sterilized for 1minute with 0.1%of mercuric chloride solution were inoculated on medium addition of 200mg/L of mancozeb as microbial inhibitors,the success rate for rescue of the contaminated explants was 40%.The sterilized explants were induction culture under semi-open environmental condition to screen the concentrations of microbial inhibitors,the results showed that the optimal induction media for five years trees was WPM medium addition of 3.0 mg/L of ZT and 0.1 mg/L of NAA and 0.1 mg/L GA₃ and 20 mg/L of mancozeb,whose inducing rate of buds and number of valid buds were 85%and 2.88,respectively,those for 430a ancient tree was WPM medium addition of 3.0 mg/L of ZT and 0.1 mg/L of NAA and 0.5 mg/L GA₃ and 100 mg/L of mancozeb,whose inducing rate of buds and number of valid buds were 96.5%and4.52,respectively.Top buds or stem segments with with axillary buds from sterile shoots of five years trees were inoculated on media for open culture to screen and optimal kind and concentration of microbial inhibitors,the results indicated that mancozeb was optimal microbial inhibitor,its optimal concentration was 50 mg/L.Top buds or stem segments with axillary buds from sterile shoots of five years trees were multiplication,the results showed that the optimal open subculture multiplication media for top buds was WPM medium addition of 3.0 mg/L of ZT and 0.1 mg/L of NAA and 0.5 mg/L GA₃ and50 mg/L of mancozeb,whose multiplication coefficient of bud was 3.5,those for stem segments with axillary buds was WPM medium addition of 1.0 mg/L of TDZ and 0.1 mg/L of NAA and 0.5 mg/L GA₃ and 50mg/L of mancozeb,whose multiplication coefficient of bud was 5.2.Stem segments with axillary buds from sterile shoots of 430a ancient tree were multiplication subculture in open,the results showed that the optimal open subculture multiplication media was WPM medium addition of 3.0 mg/L of ZT and 0.1 mg/L of NAA and 0.1 mg/L GA₃ and50 mg/L of mancozeb,whose induction rate of buds and multiplication coefficient of bud were 100%and 3.35,respectively.The clustered shoots induced in 430a ancient tree to strengthening culture,the results showed that the optimal media for strengthening culture for ancient tree was WPM medium addition of 0.5 mg/L of ZT and 0.1 mg/L of NAA and 0.1 mg/L GA₃ and 50 mg/L of mancozeb.