臺灣博碩士論文加值系統

石蒜 ( Lycoris spp. ) 為相當具發展潛力之夏季球根花卉，幼年期長達5至6年且成年母球自然分球效率僅1.8倍，因此必須建立微體繁殖系統以供新雜交選系之推展。本試驗參試種間及所育成MT-A雜種選系，探討不犧牲母球之加速繁殖技術、建立石蒜幼小花起始之再生系統，比較種間及組織部位再生能力差異性、影響不定芽發生因子、重複增生技術及評估繁殖效率，進而縮短育種年限。
幼花培養，需以生長素組合BA 10 mg/L以誘導出大量不定芽。參試幼小花不同部位組織接種於含NAA 10 mg/L及BA 10 mg/L培養基，以花梗較具再生活力，其次為花被，子房較差，舉換錦石蒜(L. sprengeri)雜交金花石蒜(L. aurea)之選系為例，不定芽再生頻率分別為83.3%、47.6%及22.2%。再生途徑方面，花梗及子房培殖體可誘導直接不定芽發生，花被則為間接不定芽發生，再生時間延緩，但其培殖體數目較多。金花石蒜與不同A核型種雜交組合選系，以帶有紅藍石蒜(L. haywardii)及換錦石蒜基因組者較具再生能力，花被培殖體再生不定芽頻率介於40-82.8%，而帶有紅花石蒜(L. radiata)基因組，再生能力普遍較低，介於8.1-46.2%。
花序衍生不定芽長成小鱗莖，加以十字分切誘導不定芽及重複繼代增生培養，將不定芽叢分切培養於NAA 2 mg/L及BA 10 mg/L之培養基中效果較好，經3個繼代週期後，總繁殖倍率可達108.5倍。體外結球及根系誘導試驗，將芽體移至液體培養基中添加蔗糖濃度90 g/L，有助小鱗莖肥大生長，2個月後小鱗莖直徑可由3 mm增大為9.3 mm。於蔗糖濃度60 g/L，液態培養基中添加KH2PO4 340 mg/L可促使蔗糖有效利用，直徑為8.6 mm，葉長可達9 cm。單獨使用NAA及IAA兩生長素對石蒜小鱗莖生長發育無顯著效應，組合NAA 1 mg/L及 BA 2 mg/L，可明顯提升葉片數至2.4枚，葉長達8.7 cm，但BA濃度大於1 mg/L根系生長被抑制。18℃下培養，鱗莖較小但可得較佳之葉數3.1枚、葉長9.3 cm及根數3.3。大小不同之小鱗莖移植出瓶，小鱗莖周徑大於2.4 cm，存活率可達100%，周徑小於2.4 cm，存活率下降至81.3%。
石蒜鱗片以2,4-D 1 mg/L及BA 5 mg/L培養8週後，可於鱗片基部產生大量癒合組織，並可產生叢生之體胚，每個培殖體平均體胚數為3.2個。以胚性癒合組織做為細胞培養之起始培殖體，空胞數量較非胚性癒合組織處理少，但尚未能培養出均質之細胞系。

The spider lily (Lycoris spp.) possesses hysteranthous and graceful flowering potential to develop as novel summer bulb plants. The juvenile period of Lycoris spp. requires 5-6 years and the natural multiplication rate is limited 1.8 times after adult stage. Thus, it is necessary to establish rapid micropropagation system benefit for the extension of new selection.
The pedicle, ovary and perianth explants were inoculated on MS medium supplemented with NAA 10 mg/L and BA 10 mg/L. The pedicle explants produced more buds than perianth or ovary. For example, the regeneration rate of adventitious bud in pedicle, perianth and ovary on selection of L. sprengeri x L. aurea were 83.3%, 47.6% and 22.2%. Direct adventitious bud formation was induced from pedicle and ovary, and indirect caulogenesis take place in perianth explant. The regeneration capacity in MT-A hybrids 2n=18(4M+3T+11A) of L. aurea 2n=14 (8M+6T) crossed with different A karyotype species 2n=22 (22A), which display higher multiplication rate between 40-82.8% in L. haywardii or L. sprengeri than 8.1-46.2% in L. radiata genome containing hybrids.
Adventitious bud originated from inflorescence grow into bulb, then it was cross cutted and cultured on the medium to induce adventitious buds. More adventitious buds formed when subcultured on the medium supplemented with NAA 2 mg/L and BA 10 mg/L. After 3rd subculture period, total regeneration rate was 108.5 times. In vitro bulbing and rooting, buds were transferred to liquid medium supplemented with sucrose 90 g/L to promote bulb growth. The diameter of bulb became 9.33 mm after culture for 2 months. Supplemented with KH2PO4 340 mg/L in the liquid medium can promote the utilizing of sucrose by bulblet. The diameter and leaf length of bulb were 8.6 mm and 9 cm. There is no significant influence on bulb growth cultured in the medium supplemented with NAA or IAA. The leaf number and leaf length of bulb were reached 2.4 and 8.7 cm when cultured in the liquid medium supplemented with NAA 1 mg/L and BA 2 mg/L. Root growth was limited when the concentration of BA higher than 1 mg/L. Bulb cultured in the 18℃ chamber has better result, the leaf number, leaf length and root number were 3.1, 9.3 cm and 3.3. Bulbs with different size were transplanted to growth chamber. The survival rate was 100% when the perimeter of bulb bigger than 2.4 cm. If perimeter below 2.4 cm the survival rate only 81.3%.
Medium supplemented with 2, 4-D 1 mg/L and BA 5 mg/L was suitable for embryo production. 3.2 embryos were obtained from each explant after culture for two months. The cells releasing from embryogenic callus is better than non-embryogenic callus, but still could not form more uniform cell line after 4 months.

目 錄
口試委員會審定書…………………………………………...……………………….Ⅰ
口試委員名錄………………………………………………..…….………………….Ⅱ
誌謝…………………………………………..………………..…...………………….Ⅲ
摘要……………………………………………… ……… …..………………………Ⅳ
Abstract…………………………………………………………..……………………Ⅵ
表目錄…………………………………………………………….…………………..XI
圖目錄……………………………………………………………..……………..…. XII
一、前言…………………………………………………………..……………………..1
二、前人研究……………………………………………………..……………………..2
(一)石蒜屬球根花卉之遺傳特點與生育習性…………………..…………………….2
1.石蒜遺傳特點…………………………………………………..…………………….2
2.石蒜生育習性…………………………………………………..…………………….3
(二)石蒜屬植物繁殖方法…………………………………………..………………….3
1.有性繁殖…………………………………………………………..………………….3
2.無性繁殖…………………………………………………………..………………….4
3.微體繁殖…………………………………………………………..………………….5
(三)誘導癒合組織形成之影響因子…………………………………..……………….6
1.培殖體………………………………………………………………..……………….6
2.培養基………………………………………………………………..……………….7
3.植物生長調節劑……………………………………………………..……………….7
4.栽培環境……………………………………………………………..……………….8
(四)癒合組織再生途徑…………………………………………………..…………….8
1.器官發生………………………………………………………………..…………….9
2.體胚發生………………………………………………………………..…………….9
3.細胞懸浮培養…………………………………………………………………..…...10
三、材料與方法…………………………………………………………………..…....11
一、器官發生……………………………………………………………………..……11
(一)幼花序培養…………………………………………………………………..…...11
1.幼花序逆分化癒合組織及再生作用……………………………..……………..….11
2.不同選系、不同培殖部位再生效率評估………………………………………..….12
(二)不定芽叢增生…………………………………………………………………...12
(三)體外結球……………………………………………………………………..…...12
1.蔗糖及KH2PO4濃度處理……………….........………………………………..…...12
2.植物生長調節劑處理…………………………………………………………..…...13
a.參試生長素NAA 0.5-2 mg/L及IAA 0.5-2 mg/L試驗……..………………….…..13
b.參試NAA 0-1 mg/L及BA 0-2 mg/L試驗…………………..…………………..….13
3.溫度處理………………………………………………………………………….....14
(四)移植出瓶……………………………………………………………………….....14
二、體胚發生…………………………………………………………………………..15
三、細胞懸浮培養………………………………………………………………..……15
四、結果…………………………………………………………………………..…...16
一、器官發生……………………………………………………………………..……16
(一)幼花序培養.............................................................................................................16
1.幼花序逆分化癒合組織及再生作用…………………………………………..…...16
2.不同選系、不同培殖部位再生效率評估……………………………………..…….16
(二)不定芽塊增生………………………………………………………………..…...17
(三)體外結球……………………………………………………………………..…...18
1.蔗糖及KH2PO4濃度處理……………...……………………………………..…...18
a.蔗糖60 g/L組合不同KH2PO4濃度處理………..……………......…………….…..18
b.蔗糖90 g/L組合不同KH2PO4濃度處理………..……..…………………..……….19
2.植物生長調節劑處理………………………………………………………..……...19
a.參試生長素NAA 0.5-2 mg/L及IAA 0.5-2 mg/L試驗……..……………….……..19
b.參試NAA 0-1 mg/L及BA 0-2 mg/L試驗…………………..………………..…….20
3.溫度處理………………………………………………………………………..…...20
(四)移植出瓶…………………………………………………………………….....…20
二、體胚發生………………………………………………………………………..…21
三、細胞懸浮培養…………………………………………………………………..…22
(1)石蒜選系(26-320)幼小花所衍生堅硬緊實、色澤金黃之非胚性瘉合組織…..….22
(2)石蒜選系(26-355)鱗片培養衍生之胚性癒合組織…………………………….....22
五、討論……………………………………………………………………………..…55
一、器官發生………………………………………………………………..…………55
(一)幼花序培養再生探討…………………………………………………..………...55
(二)不同雜交選系、不同花序部位再生能力比較………………………..………….55
1.不同花序部位再生能力…………………………………………………..………...55
2.不同雜交選系再生能力…………………………………………………..………...56
(三)鱗片誘導不定芽及再生探討……………………………………………..……...56
(四)體外結球及小植株建立…………………………………………………..……...57
二、體胚發生探討……………………………………………………………..………59
三、細胞懸浮系統……………………………………………………………..……....59
四、石蒜再生效率評估………………………………………………………..………60
六、參考文獻…………………………………………………………………..………62




表目錄
表 1. NAA或2,4-D對石蒜雙核型選系(LA x LRM1-5)花被培殖體癒合組織及器官發生之影響。......................................................................................................23
表 2. NAA及BA組合對紅花石蒜(Lycoris radiata)花被誘導再生之影響。............24
表 3.石蒜雜交選系幼小花不同部位培殖體之癒合組織及不定芽發生頻率(培養2
個月後)。............................................................................................................25
表 4.石蒜雜交選系幼小花不同部位培殖體之癒合組織及不定芽發生頻率(培養6
個月後)。............................................................................................................26
表 5. NAA及BA組合對石蒜(E-3-3 x LRK4)不同繼代週期不定芽增殖之影響。..27
表 6.蔗糖與KH2PO4濃度組合對石蒜(E-3-3 x LRK4)小鱗莖生長之影響。............28
表 7. NAA或IAA處理對石蒜(E-3-3 x LRK4)小鱗莖生長之影響。........................29
表 8. NAA與BA濃度組合對石蒜(E-3-3 x LRK4)小鱗莖生長之影響。..................30
表 9.溫度與蔗糖處理對石蒜(E-3-3 x LRK4)小鱗莖生長之影響。...........................31
表10.石蒜(E-3-3 x LRK4)不同周徑小鱗莖移植出瓶8週後存活率。.......................31
表11. NAA、2,4-D及BA不同濃度處理對石蒜(E-3-3 x LRK4)小鱗莖分切鱗片培養癒合組織、不定芽與體胚發生之影響。........................................................32








圖目錄
圖 1.石蒜組織培養參試雙核型雜種選系花序型態。……..…………..………….....33
圖 2.石蒜雙核型雜交選系[26-320(LH x LSM3)]幼小花子房、花梗及花被培養不定芽及癒合組織生長情形。……………....………………………..…………....35
圖 3.石蒜雜交選系幼花被逆分化及再分化多數不定芽之情形。.............................36
圖 4. NAA及BA組合對石蒜雙核型雜種(E-3-3 x LRK4)不定芽增殖及地上部生長之影響。……………………………….…………………………..……………38
圖 5.石蒜雙核型雜交選系(E-3-3 x LRK4)芽塊分切繼代培養誘導叢生不定芽。...40
圖 6.石蒜不定芽體外結球不正常生長情形。…………………………………….....42
圖 7. BA處理對石蒜不定芽微體培養葉片生長及抑制發根情形。…………….....43
圖 8. NAA與IAA對石蒜(E-3-3 x LRK4)小鱗莖誘導瓶內發根之影響。………....45
圖 9.石蒜(E-3-3 x LRK4)不定芽於不同蔗糖濃度及溫度下，液體培養8週後之生
長發育情形。…………………………………...…………………………...…46
圖10.石蒜雙核型雜種之小植株移植生長情形。(2個月後).......................................47
圖11.小植株於生長箱栽培2個月後根系發育正常。.................................................47
圖12. 2,4-D 1 mg/L組合BA 2、5或10 mg/L對石蒜(E-3-3 x LRK4)鱗片培養之生長發育情形。......................................................................................................48
圖13. BA不同濃度組合2,4-D 2 mg/L對石蒜(E-3-3 x LRK4)小鱗莖分切培養不定
芽及體胚發生之影響。......................................................................................50
圖14.石蒜小鱗莖分切培殖體逆分化-再分化之不同發育期體胚發生。...................52
圖15.非胚性與胚性癒合組織於液體培養基中細胞釋放比較。................................53
圖16.石蒜( E-3-3 x LRK4 )體外再生流程。................................................................61

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