應用早期篩檢蝴蝶蘭V3品系花梗莖頂組織所誘導並配合藥劑處理之擬芽球體株系達到去除病毒感染之研究

Elimination of Odontoglossum ringspot and Cymbidium mosaic viruses from Phalaenopsis hybrid "V3" through early selection of shoot-tip induced protocorm-like body line in combination with chemotherapy

簡愷彣(Kai Wen Chien)

指導教授 ： 蔡新聲 ； 張清安

朝陽科技大學/理工學院/應用化學系生化科技碩博士班/博士(2014年)

摘要

蘭花是台灣最重要的園藝產品，目前以出口為生產導向的蘭花種類超過5個以上，其中以蝴蝶蘭最知名而為台灣蘭花產業的代表。組織培養繁殖之分生瓶苗及不同大小之中小苗為目前台灣蝴蝶蘭外銷之主要項目，因此確保種苗之健康無病遂成為台灣蝴蝶蘭產業最重要之課題。過去產業發展過程中台灣蝴蝶蘭種苗之高病毒感染率曾是下游買方抱怨之項目，但近年來在產官學界之努力下，此項缺失已獲得明顯改善。蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)及齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV)被公認為具全球分佈最廣造成經濟損失最鉅的兩種病毒，台灣蝴蝶蘭也不例外。尤其有許多優良商業品種幾乎全數遭到此二病毒之感染，在無病毒種源無從尋覓之情況下，所繁殖之分生組培苗也全面感染病毒而影響其銷售。過去文獻上已累積相當多針對已感染病毒之植物進行去病毒處理(virus elimination)以重新獲得無病毒感染種源之報告與經驗，但是此等文獻迄今均未能轉化成可商業實際應用之穩定作為，究其原因應在於多數已記載之去病毒方法均落於時間耗費極長，且無法肯定獲致預期結果之格局。源此，本研究重新檢討過去累積之經驗，以蝴蝶蘭最具代表性之大白花V3品系(Phal. Yukimai×Phal. Taisuco Kochdian)為材料，採取三種不同策略比較其去病毒之效果。第一項策略乃切取病株之花梗芽莖頂組織，培養於經本研究測試且優化之擬芽球體(PLB)誘導培養基(內含10 g/L 蔗糖、0.9% (w/v) Bacto agar的1/2 MS基礎鹽類)中，經12-16週培養可穩定獲得擬芽體，誘導率約為17 %。後續將所誘導之擬芽球體單一挑出，給予特定編號之株系後對半分切，再培養於經優化後之繼代培養基 (1 g/L花寶1號培養基內含10 g/L 蔗糖、60 g/L 馬鈴薯泥、2.5 g/L 活性碳、0.5 g/L tryptone及0.9 % (w/v) Bacto agar)中進行增殖，每二個月繼代增殖一次，共至少五次繼代後再以相同培養基誘導再生為小苗。過程中每一繼代增殖之PLB株系均經過ELISA及RT-PCR檢測確認病毒感染狀態。結果發現在最原始所獲得的32個株系中有18個經過五代增殖與檢測均未發現病毒感染，其所誘導再生之小苗，也經ELISA證實為無病毒感染。過程中也發現，部分株系在第一代增殖時並未檢出病毒感染，但當其第二代增殖時卻發現有病毒感染情形，此感染情形於第三代繼代增殖後即未再出現。此一策略最終結算之去病毒比率約在56 %，獲得無毒苗時間估計至少需要10個月。第二項策略乃直接由取得之感染病毒PLB培植體挑取100個單一PLB株系進行繼代增殖，以相同之方式進行至少五次之繼代增殖，每代株系均經ELISA與RT-PCR之病毒檢測確認，最後發現有12個株系經過五代之增殖與檢測後，仍然維持無病毒狀態，其再生之小苗也檢測確認無病毒感染。過程中我們也證實如同前一策略般，有部分檢測為無病毒感染之第一代繼代株系，在第二代增殖時再被檢出病毒感染情形。相同的，此一病毒感染情形在第三代增殖後即未再出現。總結此項策略約可由增殖的PLB後代中獲得約20 %去病毒比率，所需時程約為6-7個月。第三項策略我們由確認感染病毒之PLB株系開始，將單一株系培養於含有抗病毒藥劑之繼代培養基中處理二個月，再移入不含藥劑之正常培養基，後續每二個月繼代一次同時進行病毒檢測，共至少進行五次繼代。本研究總共測試Ribavirin、Amantadine、Tamiflu、Berberine、Quercetin、牛樟芝、IMPDH抑制劑等業界常用或具有抗病毒潛力之藥劑或天然試劑，結果選出Ribavirin 及Amantadine二種進行進一步測試。我們先以感染單一病毒之PLB為材料測試上述兩藥劑在不同濃度下的去病毒效果及對PLB增殖的影響。結果發現當藥劑濃度提升時，PLB之增殖即相對受到抑制。而去病毒效果則與藥劑濃度之增加沒有對應之提升關係。兩種藥劑均在特定但並非最高之測試濃度下顯現最佳之去病毒效果。另外我們也確認Ribavirin對於ORSV與CymMV之抑制效果並無明顯差異，但Amantadine則對ORSV有明顯抑制效果，卻對CymMV完全沒有抑制能力。接著我們以複合感染ORSV及CymMV 之PLB株系為材料進行測試，若以不同濃度之單一藥劑進行處理時，所得之去病毒比率均在10 %以下。但若以上述兩藥劑搭配不同濃度組合處理時，我們發現獲得去病毒PLB株系之比率可大幅提升，其中有七種濃度組合之去病毒比率可以穩定超過30 %，甚至有一組合可以高達45 %。此一結果有別於過去文獻上之蘭花去病毒研究，首次採用Ribavirin 及Amantadine不同濃度混和處理，並且確認其可以穩定獲致30 %以上之去病毒比率，較之過去常用之單一藥劑處理之去病毒效果有明顯之提升，此一策略流程所需時間基本上與第二策略相同，但其去病毒比率卻有明顯之提升。上述三種策略進行過程中我們一再發現PLB株系在增殖初期會有檢測不到病毒，卻於下一代增殖後再被發現仍屬病毒感染之情形，因此我們確認不管PLB株系來自於莖頂組織之誘導或者來自抗病毒藥劑之處理，必須至少經過三代繼代培養與檢測仍無法檢出病毒時，方能確定為無病毒感染狀態，而可繼續維持至再生小苗。此一發現與作法有別於過去文獻有關去病毒研究時僅仰賴單一次病毒檢測即判定是否為成功去病毒之慣例，應能對未來去病毒技術在蘭花產業上之實務應用有所助益。

關鍵字:蝴蝶蘭、去病毒、擬芽球體、東亞蘭嵌紋病毒、齒舌蘭輪斑病毒、間接酵素連結免疫吸附反應、反轉錄聚合酶連鎖反應、莖頂組織

關鍵字

間接酵素連結免疫吸附反應 ； 齒舌蘭輪斑病毒 ； 東亞蘭嵌紋病毒 ； 擬芽球體 ； 去病毒 ； 蝴蝶蘭 ； 反轉錄聚合酶連鎖反應 ； 莖頂組織

並列摘要

Phalaenopsis is one of the most popular orchid plants in the global market. Due to its high economic value, a large number of hybrids with attractive combinations of spray length, bud number, flower color, fragrance, seasonality, and compactness have been developed. Several viruses have been reported to negatively impact its growth, yield and quality. Taiwan is one of the leading Phalaenopsis producing countries in the world. Phalaenopsis Sogo Yukidian “V3”, a hybrid of Phal. Yukimai × Phal. Taisuco Kochdian, characterized by its large and white-colored flowers, is a popular commercial cultivar in the market. However, “V3” hybrids are severely affected by a mix infection of Odontoglossum ringspot virus (ORSV, Tobamovirus), and Cymbidium mosaic virus (CymMV, Potexvirus). These two viruses adversely affect the growth, vigor and market value of Phalaenopsis. In recent years, Taiwan’s Phalaenopsis industry has significantly suffered from the high virus incidence rate. Therefore, aim of the present study was to investigate and develop an effective protocol to eliminate these critical viruses from Phalaenopsis Sogo Yukidian “V3” hybrid.
Phalaenopsis Sogo Yukidian “V3” hybrid plants, naturally infected with ORSV and CymMV, and protocorm-like bodies (PLBs) were procured from the Yu-Pin Biological Technology Co., Ltd., Chiayi County, Taiwan. These were used as plant materials to initiate in vitro cultures. The indexing of viruses in the plants and PLBs was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR) developed by our laboratory.
Three different strategies for an effective virus elimination in Phalaenopsis Sogo Yukidian ‘V3’ were followed. In the first strategy, shoot-tips obtained from flowering stalk of infected Phalaenopsis Sogo Yukidian ‘V3’ plants were used as explants for induction of protocorm-like bodies (PLBs). After several experimental trials, the induction of PLBs in shoot-tips was achieved on 1/2x Murashige and Skoog’s basal medium supplemented with 1 % sucrose and 0.9 % Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1 % sucrose, 6 % potato pulp, 0.5 g/L tryptone, 0.25 % activated charcoal and 0.6 % Bacto-agar. Induction of a total 32 protocorm-like bodies (PLBs) in 17/100 (17%) shoot-tips was observed between 60-90 days of culture. All the remaining explants turned brown, became necrotic, failed to grow and subsequently died within 7-14 days. These 32 PLBs were maintained as separate lines and were indexed for ORSV and CymMV viruses by ELISA and RT-PCR techniques. After first subculture, out of 32 PLB lines, 10 were found to be positive for both the viruses. In the second subculture, of the remaining 22 virus-negative lines only 4 of them showed recurrence of the two viruses. Thus, there was a re-occurrence of virus infection in the 1st and 2nd subculture, hence, at least 3 subcultures were necessary to be sure that the cultures are free of viruses. Infected PLBs were discarded. Non-infected PLBs grew into shoots, which induced roots and developed into plantlets in the same culture medium after 90-180 days.
In the second strategy, PLBs procured as aseptic cultures from the orchid company were cultured on the Hyponex medium. Of the 100 PLBs cultured, 60 survived on the culture medium and they were then singly separated and cultured. When these 60 PLB lines were indexed by Indirect-ELISA and RT-PCR for ORSV and CymMV infection, 41 lines showed the presence of both viruses. RT-PCR gel electrophoresis also showed the presence of two amplicons representing both the viruses. The remaining 19 PLB lines were all negative in both ELISA and RT-PCR tests, suggesting they were free of virus infection. However, after the following subculturing, 7 of these 19 PLB lines were confirmed to be infected, and only 12 PLB lines were free of virus, indicating a recurrence rate of 36.8 % (7/19). At the 3rd subculture, all the 12 PLBs were free of both the viruses. In both the strategies, we did not find any virus recurrence in PLBs from 3rd subculture onwards and until 7th (the last indexing), indicating that both the viruses have been totally eliminated.
In the third strategy, we used chemotherapy or anti-viral drugs to eliminate the two viruses. Several experimental trials were carried out with PLBs to evaluate the effectiveness of Ribavirin, Amantadine, Tamiflu, Berberine, Quercetin, Antrodia cinnamomea, IMPDH inhibitors commonly used in the industry to eliminate the viruses. These drugs were incorpotrated in the culture medium for 2 months. After that PLBs were transferred to medium devoid of these drugs. Out of all the anti-viral drugs, Ribavirin and Amantadine were found to be the most effective chemicals to eliminate the viruses. However, degree of effectiveness on the two viruses varied depending upon the type and concentration of the drug. Ribavirin and Amantadine were more effective when combined together and could eliminate viruses in 45% of PLBs.
The plantlets derived from PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture raised Phalaenopsis plants in greenhouse tested negative for the two viruses. Thus, in the present study, we have described an effective and practical in vitro shoot tip culture in combination with virus free PLBs selection and chemotherapy protocol for the production of Phalaenopsis hybrid “V3” plants free of ORSV and CymMV viruses. The results would be of immense help to Phalaenopsis industry in elimination of these critical viruses.

Keywords: Phalaenopsis, virus elimination, protocorm-like bodies (PLBs), Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Indirect-ELISA, RT-PCR, Shoot-tip culture.

並列關鍵字

Indirect-ELISA ； Odontoglossum ringspot virus (ORSV) ； Cymbidium mosaic virus (CymMV) ； protocorm-like bodies (PLBs) ； virus elimination ； Phalaenopsis ； RT-PCR ； Shoot-tip culture

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