紅龍果病毒性病害之研究與健康種苗生產系統之研發

Abstract

紅龍果(pitaya, dragon fruit)為近十年在臺灣興起的熱帶果樹之一，其具有高經濟價值，也不斷有新的品種推陳出新。但在前幾年的田間調查研究中，發現田間幾乎所有的紅龍果植株皆受到病毒的感染。推斷是由於現今紅龍果多以扦插的方式進行大量繁殖，若扦插母本植株帶有病毒，則易使病毒於田間迅速的擴展。為了保留罹病母本植株優良的園藝性狀，本研究嘗試建立紅龍果的植株脫毒技術，以獲得無病毒的健康苗。在進行脫毒處理之前須先建立紅龍果組織培養條件，測試出適合白肉紅龍果莖頂組織與大量繁殖之培養條件。實驗中發現由於田間紅龍果材料肉質莖的刺座絨毛與表皮角質層附著許多真菌污染源，以一般消毒方式不易去除，但由於需要大量試驗材料，後續實驗以實生苗取代。在大量繁殖的部分，將白肉紅龍果實生苗之莖段培養在含有1.5 mg/L zeatin之全量MS培養基中，誘導刺座芽體生長率為57.8%，每個刺座平均可長出1.2個芽體，且新芽平均長度為1.33公分，相較6-benzylaminupurin (BAP)有較佳的生長情形；且同濃度之zeatin也具有誘導發根的效果，因此認為白肉紅龍果莖段的培養不需經根系誘導之處理。而在莖頂組織培養部分，則是將莖頂組織培養於含1~1.5 mg/L BAP之全量MS培養基，皆有良好的芽體再生誘導效果。確認了適合紅龍果生長之培養條件後，進行植株脫毒的方法建立，本研究嘗試以玻璃化(vitrification)冷凍處理法(cryotherapy) 對紅龍果罹病株進行脫毒處理。將紅龍果實生苗莖頂組織預培養在含0.3 M蔗糖之全量MS培養基一天；以LS於室溫處理60分鐘後；接著以預冷之PVS2於冰上處理90分鐘；置換新的PVS2後，迅速丟入液態氮中處理至少60分鐘，回溫回復生長於含有1.5 mg/L BAP之全量MS培養基，可獲得大約60%之存活率與40%的再生率，確定玻璃化冷凍治療法在紅龍果上可行，但脫毒效率則需更進一步的測試。此外，本研究結果顯示感染紅龍果之病毒不會經由種子傳播；因此將種子清洗乾淨，避免發芽過程有傷口造成，即可獲得健康實生苗。為了研究紅龍果病毒造成之影響，本研究也進行了蟹爪蘭X病毒(Zygocactus virus X, ZyVX)之感染性選殖株之構築，從罹病紅龍果中選殖出p35S-ZyVX-6選殖株，經過全長序列的解序及分析，發現和前人所發現ZyVX-P39與國外發表之ZyVX-B1的RdRp與CP的胺基酸序列皆有90%以上的相同度；經接種於白藜植株，證實p35S-ZyVX-6有系統性的感染能力。以上研究對紅龍果健康種苗系統進行了初步的建立，期望未來確定其脫毒效率後，可得到健康植株，並以大量繁殖方式得到帶有優良性狀紅龍果的幼苗，改善現存於臺灣紅龍果之病毒性病害的現況，有助於紅龍果的生產與外銷。

Pitaya, also called dragon fruit, a kind of tropical fruits, has become important fruit crop in Taiwan within a decade. In our recent study, almost all of pitaya plants in the field are found infected by viruses, including Cactus virus X, Pitaya virus X and Zygocactus virus X. Pitaya is mainly propagated by cutting in Taiwan. If mother plants are infected with virus, the viral disease would spread very quickly in the field. In order to preserve the good horticultural traits of virus-infected mother plants, we planned to develop the virus elimination techniques to produce virus-free pitaya seedlings. Before executing virus eradication, the tissue culture process for pitaya needs to be set up. Because the villi near the areoles and the epidermal cuticle of pitaya stem frequently contain fungi that are difficult to remove by general disinfection methods, we decided to use white flesh pitaya seedlings in this study. When the pitaya stems with areoles were cultured on MS medium containing 1.5 mg/L zeatin, the shooting rate of 57.8%, average 1.2 shoots per areole and the mean shoot length of 1.33 cm were observed after 8 weeks. According to our results, zeatin could induce shoot more efficiently than 6-benzylaminopurine (BAP). Since new roots grew under the same concentration of zeatin, the root induction for pitaya stem culture by auxin seems unnecessary. In shoot tip tissue culture experiments, those tissues kept in MS medium containing 1~1.5 mg/L BAP all produced new shoots with good quality. Next step in this study was to eradicate virus from pitaya by vitrification cryotherapy and shoot tip tissue culture. The process of vitrification cryotherapy are as following: the shoot tips of pitaya seedlings were precultured on 0.3 M sucrose-preculture MS medium for 1 day, treated by LS for 60 min, replaced by pre-cooled PVS2 for 90 minutes on ice, refreshed by pre-cooled PVS2, put into liquid nitrogen for 60 min, then thawing and recovery culturing on MS medium containing 1.5 mg/L BAP. There were about 60% survival rate and 40% regrowth rate of the treated pitaya shoot tips and this result confirmed that vitrification cryotherapy can be applied to pitaya. Moreover, our experimental results indicated that the potexviruses which infect pitaya were not transmitted by seeds. Therefore, virus-free seedlings can be obtained by cleaning up the seeds and avoiding injury during seed germination. Finally, in order to study the effect of viruses on pitaya, we constructed the infectious clone of Zygocactus virus X (ZyVX) from the infected pitaya. After the full-length sequence of p35S-ZyVX-6 was determined and compared with the published sequences of ZyVX-P39 and ZyVX-B1, it is shown the identity of amino acid sequence in RdRp and CP genes are both above 90%. The inoculation of p35S-ZyVX-6 on Chenopodium quinoa confirmed its ability to cause systemic infection. In this study, we set up the tissue culture and vitrification cryotherapy process of pitaya, and these methods will soon be tried to eradicate the viruses from infected pitaya. Furthermore, we hope to get virus-free seedlings with good horticultural traits by mass production. Hopefully, the results of our study can reduce the pitaya virus infection and benefit the production and export of pitaya in the future.