| Villous amomum fruit, one of the valuble Chinese herbs in South China, is a useful medicine included in the 2010's Pharmacopoeis of People's Repoblic of China, and it origin from family Zingiberaceae, Amomum villosum Lour., Amomum villosum Lour.var,xanthioides T.L.Wu et Senjen and Amomumlongiligulare T.L.Wu,In Tradicional Chinese Medicine(TCM), it is used for dissipating dampness and appetizing, warming spleen and stopping diarrhea, regulating Qi flow and preventing miscarriage. Among the three kinds of herbs resources, Amomum villosum Lour.is the best. The growth of AV needs special ecological environment and it is hard to be pollinated by itself, which leading to a low yield.1 ObjectiveStudying on tissue culture, plantlet regeneration, and radiation breeding of Amomum villosum, the ideal regeneration system of Amomum villosum. was established and using adventitious buds,which got from tissue culture, for mutation breeding by radiation to explore the radiation dose and selecte the useful radiation lines. For another hand, using seeds and buds for mutation breeding by radiation to explore the radiation dose and selecte the useful the radiation lines. The purpose of this study is to find a useful mutants of Amomum villosum.2 Methods2.1 research on three kinds of cultivars of Amomum villosum seedsTo explore the differences of the shapes of seeds and germination characters of three kinds of cultivars. Characters of the seeds'appearance were observed. The morphology, color and luster, scent, one thousand-seed weight, moisture content, ripeness, plumpness, viability, germination percentage and germination vigor etc. were measured. 2.2 study on Amomum villosum seed germinationThe seeds of Amomum villosum were treated with different illumination condition, germination bed and temperature to find a optimum germination condition and using GA3 with different concentration to accelerate germination.2.3 study on Amomum villosum in vitro and it's rapid propagationAfter seeds of Amomum villosum germinating in sterile conditions, taking the basal internode of seedlings as explants, adventitious buds multiplication of Amomum villosum were studied. It was fund a way to establish a regeneration system.2.4 studies on radiation breeding of Amomum villosumThe seeds, rhizomatic buds, cultured buds of Amomum villosum were radiated by 60Co-γray to discuss the optimum irradiation dose. The different irradiation doses had some effect on the seedling growth of Amomum villosum, and to find the survival mutants.2.5 identification of the mutants by inter-simple sequence repeat (ISSR) markersAnalysis of the survival mutants was done with DNA extraction; polymerase chain reaction (PCR) amplification using 9 polymorphism primers.The ISSR molecular marker technique was applied to analyze the genome DNA diversity of radiation treated plants, which expressed no difference with control by characters of the lines'appearance.3 Results3.1 research on three kinds of cultivars of Amomum villosum seedsThe longitudinal diameter, transverse diameter, thickness, seed characteristics, germination of the three kinds of cultivars of Amomum villosum seeds were measured. The kind of ChangGuo and YuanGuo just has little difference but ChunXuan has significant difference in longitudinal diameter, one thousand-seed weight, moisture content, ripeness, plumpness, viability. Study on seed germination experiment, ChunXuan is especial, which has low germination potential and high germination rate. Above all, the kind of ChangGuo was selected for the next germination study.3.2 study on Amomum villosum seed germinationThe results showed that light is necessary for Amomum villosum seeds germination.The germination rate and germination potential of the seeds on moist filter paper are both higher than that on the MS medium.The optimal temperature for the seeds germination is 25-30℃. GA3(400 mg·L-1) priming accelerates seeds germination significantly and enhances germination potential.3.3 study on Amomum villosum in vitro and it's rapid propagationTaking the basal internode of seedlings as explants, the best initiation multiplication medium was MS+6-BA 4.0 mg·L-1+NAA0.1 mg·L-1. Shoot sprouting was initiated after 4 days, and it got 1-4 shoots after 30 d later. Besides, in the medium of MS for plant regeneration was apt.3.4 studies on radiation breeding of Amomum villosumThe result showed that the optimum irradiation dose for seeds' radiation is 13-15 Gy and for rhizomatic buds is 15-30 Gy. Presently there were 1 line, marked" 0704YG1" which grew up from seeds treated by 60Co-y ray and 7 lines, marked"0708CG1,0809CG1～0809CG6"which grew up from rhizomatic buds treated by 60Co-γray. But they didn't have significant morphological characters. Cultured buds treated with 16 Gy by 60Co-y ray has some character variations.The optimum irradiation dose for cultured buds' radiation need next explore.3.5 identification of the survival mutants by inter-simple sequence repeat markersUsing 9 primers in PCR and sepharose electrophoresis. The DNA fragments were obtained and their sizes were from 250～5000 bp through ISSR amplification, but has no polymorphic bands.4 ConclusionThe optimum germination condition for seeds of Amomum villosum germinating is found. The method to make Amomum villosum in vitro and rapid propagating is found. Basis in them, the seeds, rhizomatic buds, cultured buds were radiated by 60Co-y ray to discuss the optimum irradiation dose and obtained some survival lines. In addition, ISSR was applicated to identification the survival mutants. All those make it possible to obtain several new lines which have higher yield and better qualities through multi-generation screening from the offspring of irradiated Amomum villosum. |
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