[Rapid Propagation in vitro of Dioscorea opposita 'Guangfeng'and Its Stomatal Observation,FCM Analysis of Chromosome Ploidy and ISSR Detection of DNA Mutation].

Objective: To study the rapid propagation in vitro of Dioscorea opposita'Guangfeng', and to observe the stomas of the transplanting plantlets and potted seedlings, to test chromosome ploidy by FCM, and to detect DNA mutation by ISSR,in order to provide the technical basis for the large-scale production of Dioscorea opposita 'Guangfeng' plantlets. Methods: The technique system of Dioscorea opposita 'Guangfeng'rapid propagation in vitro was established and optimized by plant tissue culture method. The parameters of transplanting plantlets and potted seedlings were studied as follows, the stomatal parameters were observed by transparent adhesive tape method, chromosome ploidy were analyzed by FCM, and DNA mutation were detected by ISSR molecular marker. Results: The technique system of Dioscorea opposite 'Guangfeng' rapid propagation in vitro was as follows, slightly woody stem segment with a bud were selected and inoculated onto MS + KT 1 mg / L + NAA 0. 2 mg / L solid culture medium and cultured in the photoperiod of 14 h / d( the temperature was( 25 ± 2) ℃ and light intensity was 1 500 ~ 2 000 Lx) after disinfected for 1 min in 70% alcohol prior to sterilized for 12 min with 0. 1% Hg Cl2,the materials were washed with sterile water for 3 times, respectively. The new bud was cut off when it grew to 2 ~ 3cm and inoculated into MS + KT 2 mg / L + NAA 0. 5 mg / L liquid culture medium and continued to culture in above culture conditions. The whole plant was formed after cultured for about 90 d. The sealing membrane was opened in transplanting, and the plantlets was still placed in above culture conditions and cultured for 2 ~ 3 d, and then the whole plant was taken out, and the culture medium washed off and then transferred into the vessel with shallow liquid MS basic culture medium and domesticated indoor. The acclimated plantlets were taken out and transplanted in the outdoor pots with the sandy soil when the new shoots grew out, and watered one time with tap water in the morning and evening per day, the survival rate reached 100%. The results of stomatal observation, FCM analysis and ISSR detection of transplanting plantlets and potted seedlings showed that the stomatal parameters, chromosome ploidy and DNA mutation of plantlets and potted seedlings had no variation. Conclusion: The results reveal that the establishment and optimization of the technique system of Dioscorea opposita 'Guangfeng' rapid propagation in vitro is feasible, and the regenerated plants do not have genetic variation which can ensure the stability of the genetic.

[Effect of PEG-6000 Simulation Drought Stress on Physiological Characteristics of Guangfeng Medicinal Yam Plantlets].

Objective: In order to provide the theoretical basis for the Guangfeng medicinal yam( Dioscorea opposita) in field transplanting, the effect of PEG-6000 simulation drought stress on physiological characteristics of Guangfeng medicinal yam plantlets was studied. Methods: Using the method of spectrophotometer,the content of total chlorophyll,soluble total sugar, soluble protein and praline,as well as the activities of SOD,CAT and POD of Guangfeng medicinal yam plantlets were tested under PEG-6000 treatment. Results: Under PEG-6000 simulated drought stress, with the increasing of drought stress and the extension of stress time, the total chlorophyll content of Guangfeng medicinal yam plantlets continued to decline, the content of total soluble sugar, proline and MDA of Guangfeng medicinal yam plantlets significantly increased, the content of soluble protein and the activities of CAT,POD and SOD of Guangfeng medicinal yam plantlets increased at first and then decreased. Conclusion: This study reveals the changes of physiological indices of Guangfeng medicinal yam plantlets under PEG-6000 simulation drought stress, which indicated that Guangfeng medicinal yam plantlets have certain drought tolerance.

[Study on Rapid Micropropagation in Vitro Technique of Guangfeng Medicinal Yam (Dioscorea opposita) Plantlets].

OBJECTIVE: In order to provide methodology reference for virus-free and germplasm conservation of Guangfeng medicinal yam (Dioscorea opposita) plantlets, rapid micropropagation in vitro technique of Guangfeng medicinal yam plantlets was studied. METHODS: Using the method of plant tissue culture, single factor test and flow-cytometry, the basic procedure of Guangfeng medicinal yam tissue culture was established and the DNA content of Guangfeng medicinal yam plantlets and its potted seedlings was detected. RESULTS: The best disinfection procedure of stems with a bud of Guangfeng medicinal yam was washed with sterile water for three times after sterilized with 70% alcohol for 20 - 30 s and then washed with sterile water for three times again after sterilized with 0.1% mercuric chloride for 10 - 12 min; The best explants of stems with a bud of Guangfeng medicinal yam was slightly woody and more mature stems witha bud; The best proliferation culture medium of stems with a bud of Guangfeng medicinal yam was MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L; The best rooting culture medium of stems with a bud of Guangfeng medicinal yam was MS + NAA 0.5 mg/L; The best culture method of Guangfeng medicinal yam plantlets was liquid culture; The best transplanting matrix of Guangfeng medicinal yam plantlets was the mixture of paddy clay and fine sand (1: 2) or the mixture of perlite and vermiculite (1: 2); The DNA content between Guangfeng medicinal yam plantlets and its potted seedlings had no significant difference. CONCLUSION: A fast and efficient micropropagation in vitro technological system of stems with a bud of Guangfeng medicinal yam is established, and the flow cytometry detect results also show the genetic stability of Guangfeng medicinal yam plantlets, whose results provide the technical and theoretical basis for the large-scale production of Guangfeng medicinal yam plantlets.

[Preliminary study of Dioscorea bulbifera plantlet microtuber in vitro induction].

OBJECTIVE: In order to provide a theoretical basis for the microtuber factory production and its germplasm resources preservation, in vitro induction of Dioscorea bulbifera plantlet microtubers was studied. METHODS: Through plant tissue culture technique and single factor experiment method, stems with buds of Dioscorea bulbifera plantlets as explants, the effects of various factors such as sugar, inorganic salt, cultivation mode, activated carbon and physiological state of stems with buds on in vitro induction of Dioscorea bulbifera microtubers were studied. RESULTS: The optimal sugar of Dioscorea bulbifera microtuber in vitro induction was 60 g/L sucrose or 90 g/L white sugar. The best inorganic salt concentration of Dioscorea bulbifera microtuber in vitro induction was MS. The best culture method of Dioscorea bulbifera microtuber in vitro induction was solid-liquid double layer culture. Activated carbon had a significant effect on Dioscorea bulbifera microtuber in vitro induction, whose optimum concentration was 0.03%. More mature the stem with buds was, the shorter the time of microtuber formation need. CONCLUSION: This experiment establishes a rapid method of Dioscorea bulbifera microtuber in vitro induction for the first time,which provides a new way for the application of Dioscorea bulbifera microtubers in agricultural production.

[Study on cryopreservation of Pueraria lobata germplasm by vitrification].

The experiment of the cryopreservation technique on Pueraria lobata showed that: At first, the aseptic explants were treated at 4 degrees C for 5 days. Then the stems with buds were cut and precultured at 4 degrees C for 1 day in 5% DMSO + 5% sucrose media. They were dehydrated with 60% and 100% PVS2 (30% glycerol + 15% glycol + 15% dimethyl sulfoxide + 0.4 mol/L sucrose) at 0 degrees C for 30 minutes respectively. At last the stems were immersed immediately into liquid nitrogen directly and conserved for 24 hours. After rapidly thawing in a water bath at 40 degrees C for 90 seconds, the stems were washed two times with MS media supplemented with 1.2 mol/L sucrose and 10 minutes each time, then transferred on the MS media supplemented with BA 2 mg/L and NAA 1 mg/l, in dark for 7 days and then in light. The survival rate was up to 57-58% . The regenerated plant of Pueraria lobata were the same as the normal in morphology.