| Based on viruses detection of cultivated and wild Pinellia ternata (Thunb.) Breit. from Zhejiang province, Sichuan province, Anhui province, and Beijing, China, rapid propagation of virus-free cultivated P. ternata and tubers formation in vitro studied.In March 2004 and 2005, Dasheen mosaic virus (DsMV) and cucumber mosaic virus (CMV) from cultivated and wild Pinellia ternata (Thunb.) Breit. collected from different areas of China was detected by DAS-ELISA. The results showed that DsMV and CMV are both main viruses infecting P. ternata in China. The infection ratio of cultivated samples by DsMV and CMV were 71.4% and 14.3% respectively for 21 samples collected from Ningbo, Zhejiang Province; 100% and 44.4% for 18 samples from Xiaoshan, Zhejiang Province; 61.9% and 33.3% for 21 samples from Hebei Province; 50.0% and 41.7% for 12 samples from Anhui Province; 16.7% and 16.7% for 12 samples from Sichuan Province; 31.3% and none for 16 samples from Beijing. And the infection ratio of 25 wild samples from different areas of China infected by DsMV and CMV were both 20.0%.In vitro cultivation of P. ternata showed the following results: (1) Different surface disinfection treatment was available for different explant. (2) Using 0.5 mm meristem tip of/! ternata as explant, MS solid medium, with 1.5 mg/L 6-BA or with 2.0 mg/L 6-BA and 0.1 mg/L NAA was suitable for the explant initiation and shoot differentiation, and MS solid medium with 1.0 mg/L 6-BA and 0.2 mg/L NAA was found to be useful for subculturing in order to proliferation and achieve large vigorous shoots. (3) MS solid medium with 1.5-2.0 mg/L 6-BA and 0-0.1 mg/L NAA or MS solid medium with 1.0 mg/L 6-BA and 0.2 mg/L NAA, petiole of P. ternata can be induced to regenerate and stimulate large axillary shoot formation. In addition, the ability of adventitious shoot regeneration was influenced by orientation, polarity and position of the explant from mother seedling. And the number of adventious shoots regenerated was as the following: Ppl/H > Ppl/Vup >Ppl/Vin and Pp3/H > Ppl/H > Pp2/H. (4) 1/2 MS basal medium with 0.2mg/L NAA and 0.5% active carbon was used to induce the formation of large healthy roots.Virus elimination was studied on virus-infected P. ternata and the following results were obtained: Meristem-tip culture combined with thermotherapy for virus elimination from P. ternata was not obviously better than that without thermotherapy, and 0.2mm meristem-tip culture was an available means for virus elimination. According to DAS-ELISA detection tests, DsMV and CMV can be eliminated. Tube cultures and young acclimatized plantlets from embryo culture were detected without the two viruses.The promotion factors for tuber formation in vitro of P. ternata was studied and the following results were obtained: 0.005 mg/L GA3 had slight effect on tubers formation in vitro, while (0.05, 0.5, 5) mg/L ABA inhibited tubers formation; (0.2, 2.0, 20.0) mg/L 9,10-Propyl dihydro-jasmonate (9,10-PDJ) had no significant effect on tubers formation in vitro. The result of orthogonal experiment indicated that sucrose and paclobutrazol (PP333) were the most effective factors to promote tubers formation in vitro, and the combination of 50 g/L sucrose, 0.5 mg/L 6-BA, 0.5 mg/L PP333, 0.05 g/L active carbon (AC) was the best combination to induce tubers in vitro. When the concentration of sucrose increased to 50 g/L or 0.4 mg/L and PP333 was added to the MS basal medium for 40 days, the diameter and the fresh weight of tubers in vitro were 1.533 cm and 0.624 g, 1.303 cm and 0.726 g, respectively.Transplantation experiment showed that virus elimination increased tuber yield, and tubers formation in vitro linked up rapid production of virus-free P. ternata in vitro. After 30 days grow in greenhouse, the fresh weight per tuber of virus-free plantlets increased by 31.22% compared with the mother petiole cultures. After transplanted in soil for 60 days in greenhouse, the fresh weight per tuber of virus-free plantlets was 0.525g, which was less than tubers in vitro induced by 0.4 mg/L PP333 or 50 g/L sucrose only for 40 days. Both the tubers in vitro stored at 4℃ up to 5 months and tube shoot with tubers in vitro can be transplanted into soil directly without acclimation, and the percent of germination (the diameter > 0.2 cm) was 100%.SDS-PAGE analysis of total proteins revealed the difference of polypeptides between virus-free tubers and mother tubers infected virus. The total proteins of all tubers changed in the same pattern. And the content of polypeptides from virus-free tubers was higher than tubers from mother petiole cultures infected virus. The content of polypeptides from tuber in vitro was higher, otherwise, the content of polypeptides increased with the increase of fresh weight of tubers in vitro.
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