- 學位論文
異戊烯轉移酶(ipt)基因轉殖青花菜之生物安全評估
Biosafety Assessment of Isopentenyltransferase (ipt)-Gene Transformed Broccoli
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摘要
在注重基因轉殖作物於環境釋放帶來效益的同時，基因轉殖作物潛在的生態風險及可能帶來的環境問題也受到相當程度的關注，而基因轉殖株之遺傳穩定及性狀是為評估首要項目。本研究材料利用異戊烯轉移酶(isopentenyltransferase, ipt)之基因轉殖青花菜(Brassica oleracea var. italica)經過4年田間性狀評估，顯示出產量性狀受環境因子的影響大過於轉殖品系間差異，基因轉殖ipt青花菜植株性狀及產量優於對照組104(CK)，與商業雜交一代品種「綠王」相當，而將花球儲存在25±2℃，轉殖系花球黃化面積達50%天數相較於非基因轉殖青花菜能延長2-3天，對於在櫥架壽命(shelf-life)方面具有實際的應用價值。 第二階段環境生態安全評估中，基因流佈(gene flow)是基因轉殖作物生態風險評估中的一個重要指標，而花粉活性與雜交稔實率是影響作物異交潛力的主要因子。基因轉殖青花菜103品系T5代為父本，與5種近緣亞種作物(青花菜雜交一代品種-綠王及同為綠王T5代104(CK)(Brassica oleracea var. italica)、白花芥藍-富悅(B. oleracea var. alboglabra)、黃花芥藍-白格林(B. oleracea var. alboglabra)、花椰菜-麗雪(B. oleracea var. botrytis) 和不同種的小白菜(B. rapa var. chinensis)、小油菜(B. rapa))進行雜交，透過分析花粉活性與雜交率對青花菜異交潛力進行評估，提供基因轉殖青花菜商業栽培隔離距離與建立共存之參考。結果顯示在一般栽培條件下，基因轉殖青花菜與其近緣亞種作物花期同步天數在2007-2008兩年間分別為3-28天及8-44天。兩種染色法及離體萌芽法分析各品種(系)親本間花粉活性，除以螢光染色法(fluorescent diacetate, FDA)檢測其間差異未達顯著水準(P＞0.05)外，以氯化三苯基四氮唑染色法(2,3,5-triphenyltetrazolium chloride, TTC)及離體萌芽法(in vitro germination)花粉活性親本間有顯著差異(P＜0.05)；經由基因轉殖青花菜103品系與其5種近緣亞種作物之人工授粉、蜜蜂授粉及田間自然雜交的稔實率調查，種內變幅各在86.0-100%、1.1-42.5% 及0-9.0%間，而屬於種間雜交之小白菜及小油菜並無發現雜交現象，本研究顯示基因轉殖異戊烯轉移酶插入對青花菜品種異交潛力的改變甚微，但仍存在著基因流佈的可能性風險。進一步探討轉殖株雜交後代植株之競爭能力，於溫室利用同為自交系基因轉殖ipt青花菜-103轉殖系與104(CK)為花粉貢獻親，分別與4種近緣亞種作物進行人工雜交，整體而言，F1後代植株性狀以103轉殖系雜交F1後代株高較104雜交F1後代為矮，其多數性狀仍在104(CK)雜交F1後代、近緣亞種及104(CK)範圍以內，性狀改變甚微，且變異程度小於104(CK)雜交後代；103轉殖系雜交F1後代花粉活性雖未高於親本，但每果莢種子數顯著高於104(CK)雜交F1後代及雜交親本，顯示基因轉殖ipt青花菜雜交近緣亞種F1後代仍具有相當的雜交優勢。 探討基因轉殖與非基因轉殖青花菜對環境微生物群落之影響中，研究發現基因轉殖ipt青花菜之土壤pH為7.2
並列摘要
While focusing on the benefits of releasing genetically modified crops to environment, there have been considerable degrees of concern for potential ecological risks of transgenic crops and environmental issues. Genetic stabilities of traits on the transgenic plants are the main goals for assessment. Three selfed T5 lines of ipt transformed broccoli (lines 101, 102 and 103) have been screened by selections for single copy insertion, desirable horticultural traits and the activity of ipt transgene. These 3 transgenic inbred lines were evaluated in field during 2004-2007 to determine their growth, yield and shelf-life after harvest, compared with the non-transgenic inbred line (104-CK) and the original parental variety Green King. For most of the vegetative growth parameters observed. Variability measured from year-to-year was larger than that from line-to-line. Inbreeding had no significant impact morphologically and yield potential of the broccoli lines. Head productions of the transgenic inbred lines 102 and 103 were comparable to the parental variety Green King, but were significantly higher than the non-transgenic inbred line 104, as more lines 102 and 103 plants produced heavier flower heads. When flower heads were stored at 25±2℃, the period required to become 50% floret yellowing were 7.5 and 8.5 d for the transgenic lines 102 and 103 respectively, compared with 5.6 d for the non-transgenic line 104, and 5.1 d for the parental variety Green King. This study confirmed that the ipt-transformed inbred lines of broccoli exhibited acceptable appearance and yields with enhanced shelf-life. Gene flow is an important index for the second phase of environmental and ecological safety assessment.. Pollens are potential carriers for genetically modified DNA sequence to other plants. For phanerogams, pollen viability and cross-compatibility are critical factors for successful outcross hybridization. In this study, ipt transformed broccoli designated as line 103 was used to investigate potential outcross by pollen viability and pollination with non-transgenic broccolis (line 104 and Green King) and other closely related varieties, such as Chinese kales (B. oleracea var. alboglabra) ‘Fu Yue’ (white flower) and ‘Bai Ge Lin’ (green flower), cauliflower (B. oleracea var. botrytis) ‘Li Syue’, ‘Pakchoi’ (B. rapa var. chinensis), and ‘Rapa’ (B. rapa). During field trial, flowering periods of transgenic ipt broccolis and non transgenic recipients were overlapping by 3-28 days in 2007 and 8-44 days in 2008. The pollen viability was determined by triphenyl tetrazolium chloride (TTC) analysis, fluorescein diacetate (FDA) and in vitro germination, respectively. Pollen viabilities by FDA showed no significant difference (p＞0.05) for transgenic line 103, non-transgenic and other tested cultivars . However, the differences of pollen viabilities were significant (p<0.05) using TTC and in vitro germination. Outcross rate was determined by the existence of ipt gene in outcross progenies analyzed by PCR. The range of fertility rates followed by artificial, insect (bee), and nature pollination were 86-100%, 1.1-42.5% and 0-9% respectively, while no hybridization was observed for ‘Pakchoi’ and ‘Rapa’ as pollen recipients. The results showed that transgenic DNA insertion did not alter potential risk for outcross among broccoli varieties. However, there was possibility of gene transfer between transgenic and non-transgenic broccoli varieties. In order to reveal the competitive advantage of F1 progenies breed from transgenic ipt broccoli crossed with four closely related varieties, 103 transgenic line and 104(CK) inbred line were used as pollen donors and hybridized with related varieties by artificial pollination. F1 offsprings from hybridization with 103 showed larger plant height, while 104 F1 hybrid offspring showed shorter. It was also found that the seed number per pod of 103 F1 hybrid was higher than 104 (CK) hybrid F1. Results showed F1 progeny from transgenic broccoli hybrid still exhibited the phenomenon of heterosis. Another concern of potential impact from transgenic broccoli is microbial communities in environment. According to our results, transgenic ipt broccoli had no significant effects on soil pH, and 103 transgenic line exhibited higher rhizosphere organic matter content on the day of transplantation. However, soil organic matter data showed no significant difference among all lines after 90 days. In 2010, total numbers of fungi and bacteria were evaluated, and inbred line 104(CK) had significantly higher than those of other lines. Moreover, almost all examined microbial populations showed significant difference among transgenic and non-transgenic lines during growing season, and there was no significant difference after harvest and tillage. Nevertheless, the populations of gram-negative bacteria, urea-oxidizing bacteria and cellulolytic bacteria showed difference on 105th days after transplanting in 2010. Similarity coefficient ofsoil microorganism were scored between transgenic and non-transgenic broccoli lines also examined. The result showed that commercial F1 hybrid variety ‘Green King’ had lower similarity coefficient score than the other two lines. Transformation of a cytokinin limiting enzyme adenosine ipt gene enhanced stress tolerance by delaying the onset of flower head wilting which prolongs the shelf-life of broccolis. Assess the effectiveness of transgenic ipt gene on improving broccoli stress tolerance and its commercialization value, transgenic broccoli was sampled by lyophilizing the 2nd leaf from top. Overall, transgenic line 103 expressed higher ipt transcripts levels than those of 102, and as a result line 103 withstood unfavorable environmental stresses better than 102 in general. Interestingly, the ipt expression levels varied over time as observed in those experiments for drought and heat treatments. Drought stress can decrease the expression levels of ipt gene but the effect of heat stress on ipt gene expression was temperature and duration specific, therefore it was inclusive. The introduction of ipt gene to transgenic broccoli line 103 enhanced the ability of water retention in leaves while ethylene production was reduced compared with transgenic line 102 or other non-transgenic lines whenever broccolis were subjected to the treatments of flood, drought, or heat stress.
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