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研究生:蘇熙倩
研究生(外文):Hsia-Chien Su
論文名稱:影響水稻再生及農桿菌轉殖發生褐化因子之探討
論文名稱(外文):Factors affecting rice regeneration and brownization of Agrobacterium-mediated transformation.
指導教授:朱德民朱德民引用關係夏奇鈮
指導教授(外文):Teh-Ming ChuChi-Ni Hsia
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農藝學系
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:84
中文關鍵詞:醣類成分與轉殖品種水稻再生抗生素菌液濃度褐化
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本研究以台農67號、台稉8號及越光三品種為材料,探討影響水稻再生及農桿菌轉殖褐化之因子,希望此一研究所建立之方法未來能應用於其他有用基因之轉殖,育成更多具有新特性之轉殖水稻。

(一) 水稻再生試驗
以成熟胚誘導癒傷組織後進行液體細胞懸浮培養再生試驗。在S1及NL兩液體培養基培養4週後發現,台稉8號於NL液體培養基有較高之鮮重;越光則以S1液體培養時較佳,台農67號於兩液體培養基中鮮重無差異。以R1M及R1兩種培養基中,繼代時分別繼代於添加10 g/l agar或3 g/l gelrite凝膠物質之再生培養基中,其中以R1M培養雞添加10 g/l agar繼代於3 g/l gelrite有最高之綠點數(44%)。不同齡期(10、20及30天)之癒傷組織於H、O及C培養系統中培養得到10﹪、6﹪及4﹪之再生率,其餘天齡在三種培養系統中均無芽體之再生。

(二) 影響農桿菌轉殖之各項因子試驗
水稻成熟胚經誘導癒傷組織後,以帶有ap1基因之pCAMBIA1304二元載體之LBA4404及EHA105農桿菌進行轉殖試驗。以誘導5天之培植體進行轉殖試驗時,經hygromycin篩選1週後,在各水稻品種及兩菌系中褐化率皆高(86.7﹪以上);以誘導3週之培植體進行轉殖試驗時,以EHA105感染台農67號褐化率最低僅7.8﹪,但以LBA4404感染越光之褐化率可達68.3﹪。利用EHA105進行不同菌液濃度感染台農67號及越光兩品種,結果顯示品種之差異性大,菌液濃度在共培養後對褐化率影響顯著,但抗生素篩選後褐化率則無差異。
以台農67號成熟胚誘導之癒傷組織利用帶有pCAMBIA1301二元載體之C58C1菌系進行一系列之轉殖試驗, OD600值在0.1-0.3之間感染15分鐘之處理褐化率最低(72﹪),以OD600值在0.7-1.0之間感染10分鐘之褐化率最高(94﹪); 感染用之AAM溶液以30 g/l山梨醇及30 g/l 麥芽糖取代10 g/l 葡萄糖處理時,培植體之褐化雖無改善,但在共培養基中添加山梨醇及麥芽糖之處理中,有10﹪之培植體可觀測到GUS基因的表現;於農桿菌感染過程於接種時期添加抗氧化物100 mg/l 半胱胺酸在hygromycin篩選2週後有最低之褐化率。添加200 μM acetosyringone於培養基中與不添加之處理相較亦無顯著之差異。以再生培養基添加或不添加ABA做為共培養基時,處理間之褐化率並無顯著之差異。培養基中添加200 mg/l timentin即能有效抑制農桿菌之生長,且對再生無不良之影響。於影響培植體褐化因子之試驗中發現培植體之褐化主要為農桿菌感染、hygromycin篩選以及農桿菌感染與hygromycin之加成作用更促進培植體之褐化。
綜合以上之試驗結果,三供試品種以台農67號品種之癒傷組織為培植體,配合適當之再生系統,以EHA105菌系OD600=0.75之濃度進行感染,AAM感染液中添加100 mg/l半胱胺酸及30 g/l山梨醇及麥芽糖均有利於農桿菌之轉殖。
Callus induced from the elite rice varieties, Tainung 67 (TNG67), Taikeng 8 (TK8) and Koshihikari (KS), were used to study factors for affecting rice regeneration and Agrobacterium-mediated transformation. Methods established from this studies may apply to transfer of other usful genes for creating transgenic rice with various agronomic traits.

Experiments for rice regeneration
Callus induced from mature seeds were used for establishing cell suspension culture and for regeneration in solid media. The structures of suspension cells induced from various varieties were different. After cultured in S1 and NL liquid medium for 4 weeks, TK8 had higher fresh weight in NL liquid medium, however the fresh weight of KS was higher in S1 liquid medium and no difference for TNG67 on both medium. In the basal medium combining gelling agent experiment, the highest green spots per treatment 44% was found from the treatment by using R1M callus inducing medium adding 10 g/l agar in combination with the regeneration medium containing with 3 g/l gelrite. Callus of 10, 20 and 30-day-old were cultured on three different media for regeneration, the highest regeneration rate was obtained from 10-day-old callus cultured and no shoot regeneration from 20 and 30-day-old callus was found.

Factors of effects on Agrobacterium transformation
Calli induced from TNG67, TK8, and KS mature seeds were infected with Agrobacterium strain, EHA105 and LBA4404, harboard with the binary vector pCAMBIA1304 containing with ap1 gene. The browning rate of 5-day-old callus after infected with both strains was higher than 86.7% for all varieties. On the other hand, the lowest browning rate of 7.8% for 3-week-old TNG67 calli after infected with EHA105 strain after hygromycin selection was abtained. However, the highest browning rate of 68.3% was also found on KS infected with LBA4404 after 1 week hygromycin selection. An experiment using 2 varieties infected by 2 Agrobacterium strains in various concentrations was conducted. The results showed that variety had significant effcet on browing rate and infection concentration of bacterium only affected browning rate after co-culture, but not after hygromycin selection.
Callus induced from TNG67 mature seeds infection with pCAMBIA1301/C58C1 was used to test several factors for transformation efficiency. The lowest browning rate, 72%, was obtained from OD600=0.1-0.3 treatment for 15 minutes, and the highest browning rate, 94%, were obtained from OD600=0.7-1.0 for 10 minutes. However, there was no significant difference among all treatments. No effect on browning prevention by adding 30 g/l sorbitol and 30 g/l maltose instead of 10 g/l glucose in AAM infection medium was found however 10% of GUS transient expression obtained in the treatment using sorbitol and maltose for co-culturing medium. Browning rate was inhibited in the treatment of adding 100 mg/l cysteine to AAM medium. No significant difference for browning prevention by adding 200 μM acetosyringone or 5 mg/l ABA to the medium was obtained. The concentration as low as 200 mg/l was effective for disinfection by using timentin as antibiotic and no inhibition effect on producing green spots. It was found that there may be synergistic effects on Agrobacterium infection and hygromycin selection on increasing of explant browning.
Conclusion with the results from these experiments, it is suggested that using the variety TNG67 which has lower browning rate as the explants, culturing in an optimal regeneration system in combination with EHA105 strain with OD600=0.75 for infection and the infection medium containing 100 mg/l cysteine, 30 g/l sorbitol, and maltose in addition for browning prevention may further improve transformation efficiency.
圖目錄-------------------------------------------------------------------------i
表目錄-------------------------------------------------------------------------ii
中文摘要---------------------------------------------------------------------- 1
英文摘要---------------------------------------------------------------------- 3
前言---------------------------------------------------------------------------- 5
前人研究---------------------------------------------------------------------- 7
一、水稻再生之研究------------------------------------------------------- 7
二、農桿菌轉殖於水稻之應用------------------------------------------11
三、影響農桿菌轉殖之因子---------------------------------------------12
四、轉殖水稻之表現------------------------------------------------------18
材料與方法------------------------------------------------------------------20
一、植物材料---------------------------------------------------------------20
二、培養基配製及培養環境---------------------------------------------20
三、農桿菌轉殖------------------------------------------------------------20
四、水稻再生試驗---------------------------------------------------------21
(一)水稻癒傷組織誘導懸浮細胞培養及其再生---------------21
(二)不同懸浮細胞培養液對懸浮細胞生長之影響------------22
(三)凝膠物質對芽體再生之影響---------------------------------22
(四)培植體齡期與再生培養基對再生之影響------------------22
五、影響農桿菌轉殖之各項因子試驗---------------------------------22
(一)培植體齡期對轉殖之影響------------------------------------23
(二)菌液濃度對轉殖之影響---------------------------------------23
(三)菌液濃度與感染時間對農桿菌轉殖之影響---------------23
(四)共培養之培養基成分對農桿菌轉殖的影響---------------24
1. 感染液及共培養培養基中醣類成分之影響-----------------24
2. 共培養基中植物生長調節劑對農桿菌轉殖之影響------- 24
3. 酚類化合物acetosyringone(AS)對農桿菌轉殖之影響-25
(五)抗氧化物對農桿菌轉殖之影響------------------------------25
(六)Timentin抗生素對殺菌及再生之影響---------------------25
(七)影響培植體褐化之因子試驗---------------------------------26
結果---------------------------------------------------------------------------27
討論---------------------------------------------------------------------------55
參考文獻---------------------------------------------------------------------62
附錄---------------------------------------------------------------------------75
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