( CymM v) Erad ica tion of CymM v from T issue Cultures of C ym b id ium sinense w ith Chem otherapy

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Θήρων Χρηστόπουλος
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1 2005, 32 (6) : Acta Horticulturae Sinica ( CymM v) ( 1,,, ; 2, ) : (CymMv) 1 2 mm, 0, mg L m in, RT2PCR : RNA PCR, 767 bp CymMv,, 7219%; 20 mg L - 1, 100%, 5 6,,, 40 mg L - 1,, : ; ; ; RT2PCR : S 68 : A : X (2005) Erad ica tion of CymM v from T issue Cultures of C ym b id ium sinense w ith Chem otherapy J iang L ing 1, Zhang M ingtao 1, Chen Zexiong 1, and Ma Guohua 2 ( 1 D epartm ent of Horticulture, Huazhong Agricultural U niversity, Key Laboratory of Horticultural Plant B iology, M inistry of Educa2 tion, N ational Indoor Conservation Center of V irus2free Germ plasm s of Fruit Crops, W uhan , China; 2 Huanan B otanical Garden, the Chinese Academ y of Sciences, Guangzhou , China) Abstract: Meristem2tip of p rotocorm s of Cym bidium sinense was emp loyed to study eradication of CymMv by combining tissue culture and chemotherapy. M eristem 2tip of p rotocorm s about 1-2 mm in length were ex2 cised and immersed separately with 0, 20 and 40 mg L - 1 of antiviral agent, ribavirin for 15 m ins, followed by the sub2culture, the regenerated p lantlets were achieved. Above materials were determ ined for virus infec2 tion by RT2PCR assay. Experiment indicated that RNA of virus2infected samp les were determ ined and the am2 p lified 767 bp specific fragments of CymM v could be visibled clearly in agarose gel electrophoresis, and no p roduct appeared in the health control; only 7219% of the p lantlets obtained merely by meristem of p rotocorm s culture were virus2free, while the percentage was 100% for p lantlets regenerated from m eristem of p rotocorm s, ever treated by 20 mg L - 1 of ribavirin, though the chem ical injured tissue to certain extents, which were still recovered, efficient p roliferation of adventitious buds were achieved, and p lantlets were regenerated subse2 quently after 5-6 months. W hen treated with 40 mg L - 1 of ribavirin following above2mentioned method, meristem of p rotocorm s becam e transparent and browned, and the cell vigor could not be recovered. Key words: Cym bidium sinense (Andr. ) W illd. ; CymMv (Cymbidium mosaic virus) ; R ibavirin; RT2PCR, 27, ( Cymbidium mosaic virus, CymMv) (Odontoglossum ringspot, ORSV) 1 CymMv,, : ; : : (200306)

2 6 : (CymMV) 1057,,,, 2 7, ;, 7,,, Stone 8 CymMv ; 2, 7, L im (Mokara Char Kuan Pink ), Toussaint Cym bidium Sw. 10, 11 9, 12, 13, RT2PCR 14 pg,, RT2PCR, 1 111, - 70, 112,, 30 m in, 70% 5 s 2 40, 2% ( mg L - 1 ) 30 m in,, 4 5, (25 1), mol m - 2 s - 1, 14 h /d, 70% 80% 113 MS + 62BA 4 mg L - 1 +NAA 1 mg L g L - 1 (ph 515),, 3 4, 1 2,, MS + 62BA 1 mg L - 1 +NAA 015 mg L g L /2MS + NAA 2 mg L g L g L % mm, mg L m in, 4, 50, 3 2 3,,,, 115 RT2PCR 14 RNA, A jjikuttira 15 CymMv, RT2PCR, CymMv ( + ) 5π2TAGCTAGGCCCCTGGC23π ( - ) 5π2CTTTAGAAAACCA2 CACGC23π RT2PCR PTC2100 Peltier Thermal Cycler (MJ Research)

3 TaKaRa One step RNA PCR Kit (AMV) RT2PCR : m in, s, 60, 015, 30 s, 72 2 m in, 10, s, s, 72 2 m in, 21, 72 7 m in PCR 2% (Mersco), 60 V 50 m in,, 2 211, 20 mg L /4,,, 4,, 30 d 1, 2,,,, d, 5 6 (,, (, 1 3) 40 mg L - 1 4),,, 4,,,,, CymMv,, d RT2PCR, (, 5) RNA, cdna, CymMv PCR,, 767 bp, ( 1) RT2PCR, CymMv RT2 PCR, 20 mg L - 1, RNA, CymMv, 2711% CymMv ( 1 ), 7219%, 20 mg L - 1 ( P < 0101) 1 CymM v Table 1 Effect on erad ica tion of CymM v from Cym bidium Treatment sinense w ith r ibav ir in trea tm en t RT2PCR 3 Ratio of virus2infected ( % ) 1 RT2PCR 1. ; 2., ; 3. RNA; mg L - 1 ; 5. DL2000 marker; 6. ; cm ; 8. 0 mg L - 1 ribavirin 13 / A F ig. 1 RT2PCR a ssays for the plan tlets of erad ica tion v irus 20 mg L - 1 ribavirin 0 /38 0B 1. Regenerated plantlets inducted from protocorn were achieved; 40 mg L - 1 ribavirin The samp le was not treated with ribavirin, regenerated plantlets Positive control 6 /6 100 from p rotocorn with invisible symp tom were achieved, and the specific Negative control 0 / bp fragments appeared; 3. Before eradication the virus, RNA : 3 RT2PCR, extracted from tight callus, the specific 767 bp fragments appeared; ; 3 3, 4. Treated with 20 mg L - 1 of ribavirin, the sample could not amplified Note: 3 Numerator expressed that the number of samp leswere de2 CymMv specific fragment; 5. DL2000 marker; 6. Possitive control; term ined with specific amp lified fragments, and denom inator was total 7. Meristem2tip of 015 cm in size for initiation culture, RT2PCR assay number of samp les, which were determined; 3 3 Becomed brown and p lantlet did not formed. exp ressed CymMv specific fragment; 8. Nagative control.

4 6 : (CymMV) ,,, ( 1 2 mm ),, 7219 %, 1 2 mm, L im 10,,, RNA,, CymMv 20 mg L - 1, 100%,,,, Klein 2,, : 1 Zettler F W, Ko N J, W isler G C. V irus of orchid and their control. Plant D isease, 1990, 74: Klein R E, L ivingston C H. Eradication of potato virus X and S from potato shoot2tip cultureswith ribavirin. Phytopathology, 1983, 73 (7) : Manganaris G A, Economou A S, Boubourakas IN, Katis N I. Elim ination of PPV and PNRSV through thermotherapy and meristem2tip cul2 ture in nectarine. Plant Cell Reports, 2003, 22: Mellor F C, Stace2Sm ith R. V irus2free potatoes by tissue culture. In: Reinert J, Bajaj Y P S. App lied and fundamental aspects of p lant cell, tissue and organ culturesed. Berlin Heidelberg: Sp ringer2verlag, Navarro L. App lication of shoot2tip grafting in vitro to woody species. Acta Hortic., 1988, 227: Shu W, Timon B. Prelim inary studies on the methods of getting virus2free peach p lantlets in vitro. Acta Hortic., 1993, 374: Yogesh P, A sha S. Elim ination of sugarcane yellow leaf virus and sugrcane bacilliform virus by tissue culture. Food and Agricultural Research Council, Reduit. Mauritius AMAS, Stone O M. The elim ination of four viruses from U llucus tuberosa by meristem2tip culture and chemotherapy. Annals of App lied B iology, 1982, 101: ,,.., 1999, 7 (1) : Chen L, Pan R C, Chen R M. Effects of media, growth regulators and dividing on the growth of Cym bidium sinense p rotocorm s cultured in vitro. Journal of Trop ical and Subtrop ical Botany, 1999, 7 (1) : ( in Chinese) 10 L im S T, Wong SM, Goh C J. Elim imation of cymbidium mosaid virus and dontoglossum ringspot virus from orchids by meristem culture and thin section culture with chemotherapy. Annals of App lied B iology, 1993, 122: Toussaint A J. Kummert C, Maroquin A, Lebrun A, Roggemans J. U se of V IRAZOLE to eradicate odontoglossum ringspot virus from in vitro cultures of Cym bidium Sw. Plant Cell Tissue and O rgan Culture, 1993, 32: ,,,,.., 1998, 18 (2) : Zeng S J, Cheng S J, Zhang J L, Zhao F B, Huang X L. A study on tissue and rapid p ropagation of Cym bidium sinense and its hybrids in vitro. Guihaia, 1998, 18 (2) : ( in Chinese) 13,,.., 2003, 16 (4) : Xiang Y, Yu F A, Peng Z H. Tissue culture of Cym bidium sinensis. Forest Research, 2003, 16 (4) : ( in Chinese) 14 J, D W :, , Sambrook J, Russell D W. Molecular cloning: a laboratory manual. 3 rd ed. Translate by Huang P T. Beijing: Science Press, , ( in Chinese) 15 A jjikuttira P A, L im2ho C L, Woon M H, Rgu K H, Chang C A, Loh C S, Wong SM. Genetic variability in the coat p rotein genes of two orchid viruses: Cymvidium mosaic virus and Odontog lossum ringspot virus. A rchives of V irology, 2002, 147:

5 : 1. ; mm, 20 mg L - 1,, ; 3. ; mg L - 1,, ; 5. Explana tion of pla tes: Erad ica te the CymM v by protocorm s culture and an tiv ira l trea tm en t in Cym bidium sinense 1. Formation of p ro2 tocorm s and adventitious buds from meristem tip of Cym bidium sinense; 2. Meristem of p rotocorm s about 1-2 mm were peeled off and treated by 20 mg L - 1 of antiviral agent ribavirin, and sub2cultured in medium containing corresponding ribavirin, which exp ressed green; 3. Induction re2 generation of the p lantlets; 4. Treated by 40 mg L - 1 of antiviral agent ribavirin inoculated in medium containing corresponding ribavirin, p roto2 corm s exp ressed transparent and brown; 5. Anomalistic, yellowish mottle symp tom appeared in virus2infected leaf of Cym bidium sinense. ( ) ( ) 144, 1 152, 120, 305, 338 ; 195, 160,, 50%,,,, : 101 ( ) ( ) 521, 389, 134, ,,,,,,,, : 69 ( ) 12,

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