Caulogenesis and Somatic Embryogenesis in Bulblet Section Culture of Spider Lily Dikaryotype Hybrids (Lycoris aurea x L. radiata.)

金花石蒜(Lycoris aurea)為原生於台灣之夏季球根花卉，其花型優美切花品質佳，且能適應台灣夏季高溫之氣候開花，屬極具發展潛力之夏季花卉。在繁殖上，因幼年期長、母球自然分球率低等因素，造成雜交選系大量繁殖推廣上的障礙。本研究主要以前人所誘導之體外小鳞莖作為起始材料，探討如何加速繁殖石蒜雜交選系。試材料為本實驗室以金花石蒜 x 紅花石蒜(Lycoris radiata)(E-3-3 x LRK4)雜交，所育成之雙核型雜種2n=18(4M+3T+11A) 。取用前人由幼花序所誘導之體外小鳞莖作為起始材料，將小鳞莖做十字分切後接種於培養基中。培養基中添加弱生長素(NAA、IAA等)或未添加生長素之處理，器官發生途徑仍然以直接發生為主，而隨著生長素的效應獲得叢生不定芽塊，並且伴隨少量癒合組織。如以MS培養基添加BA及IAA各2 mg/L培養，不定芽發生率達60%，而癒合組織發生率僅11%。使用強效生長素(2,4-D、Picloram、Dicamba)處理，器官及體胚發生途徑為間接再生。如以MS培養基添加IAA或NAA 2 mg/L組合BA 2 mg/L以及2,4-D 1 mg/L，癒傷組織發生率可達40%、體胚發生率為34%。若將基本鹽類換成N6培養基，組合BA 2 mg/L及2,4-D 1 mg/L，癒傷組織發生率可達100%，且體胚發生率達40%，能獲得較多胚性癒傷組織。光照處理對於石蒜鱗片誘導逆分化及再分化發生率，均高於培養於黑暗環境下，但未達顯著差異。根據結果顯示，石蒜雙核型雜交選系體外小鳞莖鱗片培養，誘導逆分化及體胚發生，使用2,4-D濃度較高(高於2 mg/L)易使體胚不正常，濃度太低(0.1-0.5 mg/L)則誘導效果較差。用短暫浸漬系統(Temporary immersion system, TIS) 加速石蒜鱗片繁殖不定芽，比較培養於固體培養基繁殖速率。使用TIS繁殖不定芽，繁殖一代所需時間可較固體培養基繁殖縮短4週。結果顯示將培植體以MS固體培養基添加NAA及BA各2 mg/L預培養4週後，再接種於以MS基本培養基添加NAA 2 mg/L及BA 10 mg/L，浸漬頻率為每三小時浸漬15分鐘，可獲得直徑大於2 mm之不定芽數目達45.3個較佳。不定芽培養於MS添加高糖(90g/L)液體培養基中4週後，即可出瓶成苗。所誘導之胚性癒合組織進行細胞懸浮震盪培養，以0.4 g左右癒傷組織，加入N6培養基添加2,4-D 1 mg/L之12 mL液體培養基中培養。經過6個月繼代，可獲得細胞外觀為沙狀，細胞組成多為細胞質濃厚、形狀為圓形或橢圓形之細胞團。胞外pH值由起始培養4.7逐漸上升至6.0-6.25。在懸浮細胞長期繼代過程中，胞外pH維持在6.25左右。而添加pH緩衝物MES (2-N-morpholino-ethanesulfonic)對於細胞生成量與胞外pH變化，以N6D1組合MES 10 g/L，pH值於滅菌前調整為6.3。經過28天培養後，每5 ml細胞液中細胞沉澱量達0.3 mL較佳，而胞外pH值則能穩定維持在6.3左右。石蒜懸浮細胞平板培養試驗，以細胞團大小大於15目，平板培養於SH培養基或BDS培養基分別添加picolran 0.03 mg/L及BA 2 mg/L，癒傷組織上大量再生表面為光滑且有光澤組織，與癒傷組織表面粗糙有所區別。但經過4-8週培養仍無法觀察小植株再生，因此如何誘導懸浮細胞朝體胚或不定芽再生，仍須進一步研究。Lycoris aurea is the native summer flowers in Taiwan. It’s beautiful cut flowers of good quality and can adapt to Taiwan’s hot weather. It’s a great potential for development of summer flowers. They require four to five years from seed to flower, and propagation rate is restrained. This study focused on how to speed up the breeding of Lycoris hybrid.Experiment material is Lycoris dikaryotype hybrid E-3-3 x LRK4. Using adventitious bud originated from inflorescence to be the initiatory explants, and cut by cross then cultured on the media to induce. Using mind auxin (NAA, IAA) or PGR-free medium induced direct organogenesis and a few callus formed. Foe example cultured on the MS medium supplemented with IAA 2 mg/L + BA 2 mg/L the regeneration rate of adventitious buds is 60%, but callus formed only 11%. Using strong auxin (2,4-D, Picloram and Dicamba ) induced indirect organogenesis and somatic mebryogenesis. Like MS medium supplemented with NAA or IAA 2mg/L+BA 2 mg/L+2, 4-D 1mg/L can induce more embryogenic callus. Dark-Light treatment is no significant impact on induced callus, and the rates are higher then 54%. More embryogenic callus formed on N6 medium with 2,4-D 1 mg/L and BA 2 mg/L. The dedifferentiation rates of callus were 100% and the regeneration rate of somatic embryo were 55%. Higher concentration (2 mg/L) of 2,4-D can make abnormal embryos occurred, and lower concentration (0.1-0.5 mg/L) induced less effective.sing temporary immersion system (TIS) speeds up the breeding of Lycoris scales adventitious buds, compared with the solid medium reproduction rate. To propagation the shoot for one generation on TIS could four-weeks-shorter then on solid medium. The but proliferation was induced on MS medium supplemented with NAA 2 mg/L and BA 10mg/L, results indicated 45.3 buds under 15 min/3hr immersion time. After culture in liquid medium with 90 g/L sucrose 4 weeks, the bulblet grew well and the survival rate of plantlet was high.n cell suspension culture, embryogenic callus cultured in liquid N6 medium with 2,4-D 1 mg/L. After 6 months subculture, the cells can be observed for the sand-like appearance, most of the cytoplasm of cells thick, round or oval shape for the shape of the cell mass. Extracellular pH change values from 4.7 start training gradually increased to 6.0-6.25.dd pH buffer effect the formation of cells and extra cellular pH change. Suspension cells cultured in liquid N6 medium with 2,4-D 1 mg/L and MES (2-N-morpholino-ethanesulfonic) 10 g/L, and controlling extracellular pH at 6.3 before autoclave. After cultured, parked cell volume is 0.3 mL better then others, and extracellular pH can maintain stability in around 6.3.ycoris suspension cell culture plate tests, the treatments including plating composed of medium, add pH buffer, MES, pretreatment cells and regeneration medium and classified according to size before plating. Only the larger cell mass (> 15 mash) can be observed that the proliferation, but not further observed that the cell mass of renewable adventitious buds or somatic embryos.口試委員會審定書…………………………………………...……………………….Ⅰ試委員名錄………………………………………………..…….………………….Ⅱ謝…………………………………………..………………..…...………………….Ⅲ要……………………………………………… ……… …..………………………Ⅳbstract…………………………………………………………..……………………Ⅵ目錄…………………………………………………………….…………………..XI目錄……………………………………………………………..……………..…..XII言(Introduction) .........................................................................................................1人研究(Literature Review) ........................................................................................2、球根花卉繁殖方式................................................................................................2一)自然分球法..............................................................................................................2二)實生苗播種法..........................................................................................................2三)鱗片繁殖法..............................................................................................................2四)微體繁殖法..............................................................................................................3、影響球根花卉微體繁殖之因素............................................................................3一)培植體來源.............................................................................................................3.胚組織培植體(Embryonic tissue) ..............................................................................3.體組織培植體(Somatic tissue) ..................................................................................3.生殖組織培植體(Reproduction tissue) ......................................................................4.變體組織培植體(Modified ploidy tissue) .................................................................4.次生培植體(Secondary explant) ................................................................................4二)基本培養基..............................................................................................................5三) 生長調節劑............................................................................................................5四)再生途徑..................................................................................................................6.器官發生(Organogenesis) ..........................................................................................6.體胚發生(Somatic embryogenesis) ...........................................................................7、建立細胞懸浮培養................................................................................................8、利用短暫浸漬系統加速再生................................................................................9.短暫浸漬系統(Temporary immersion system)簡介...................................................9.短暫浸漬系統影響培植體生長之探討.....................................................................10料方法(Materials and Methods) ...............................................................................11、參試材料................................................................................................................11、試驗方法................................................................................................................11一)起始培植體來源及培養環境.................................................................................11二)誘導石蒜雙核型雜種逆分化、體胚再生及器官發生.........................................11.組合生長調節劑對逆分化、不定芽以及體胚發生之影響.....................................11.光照、2,4-D濃度與內外鱗片處理...........................................................................11.基礎鹽類培養基MS、N6及B5處理......................................................................12.N6培養基組合不同生長素處理...............................................................................12三)試驗建立石蒜雜交選系細胞懸浮系統之條件.....................................................12.試驗建立細胞懸浮系統所需培養基條件.................................................................13.細胞接種量對於石蒜懸浮細胞生長發育因子試驗.................................................13.繼代間隔對於石蒜懸浮細胞生長量之試驗.............................................................13.添加pH緩衝物質處理..............................................................................................131)試驗pH緩衝物質添加量.........................................................................................132)試驗調整pH值之影響.............................................................................................143)比較增生培養基(N6D1)與再生培養基(SH3)添加pH緩衝物質之處理...............14.胚苗轉換.....................................................................................................................15.平板培養.....................................................................................................................151)試驗材料取用由不同起始釋放細胞培養基...........................................................152)細胞團大小及培養基處組成對於平板培養之影響...............................................153)細胞前處理對於平板培養之影響...........................................................................15五)利用短暫浸漬系統加速不定芽塊增生.................................................................16.固體培養及使用短暫浸漬統對不定芽再生數量及速率之影響。.........................16.前處理、BA濃度及浸漬頻率對短暫浸漬系統再生不定芽之影響。...................16.處理浸漬頻率對不定芽塊再生之影響.....................................................................16六) 不定芽、擬胚以及胚性癒合組織之切片解剖觀察...........................................17果(Results) ................................................................................................................18一)石蒜雙核型雜種小鳞莖分切培養與再生.............................................................18.不同生長素組合細胞分裂素對逆分化、不定芽及體胚發生之影響.....................18.光照、2,4-D濃度處理...............................................................................................19.基礎鹽類培養基MS、N6及B5處理......................................................................19.N6培養基組合不同生長素處理...............................................................................20二)石蒜雜交選系細胞懸浮培養系統之條件.............................................................20. 基本鹽類配方對於細胞懸浮培養之影響............................................................20. 細胞接種量對於石蒜懸浮細胞生長發育因子試驗............................................22. 繼代間隔對於石蒜懸浮細胞生長量之試驗........................................................22. 觀察不同pH下細胞情形.....................................................................................22. 添加pH緩衝物質處理.........................................................................................221) 試驗pH緩衝物質添加量....................................................................................222) 試驗調控pH值對細胞生成之影響....................................................................233) 比較增生培養基(N6D1)與再生培養基(SH3)添加pH緩衝物質之處理.........24三) 胚苗轉換與平板培養...........................................................................................24.胚苗轉換.....................................................................................................................24.平板培養 ...................................................................................................................251)試驗材料取用由不同起始釋放細胞培養基...........................................................252)細胞團大小及培養基組成對於平板培養之影響...................................................253)細胞前處理對於平板培養之影響...........................................................................26四)利用短暫浸漬系統加速不定芽塊增生.................................................................26.比較固體培養及再生不定芽塊................................................................................26.試驗BA濃度 2、5、10 mg/L及前處理對不定芽再生之影響............................27.浸漬頻率對於不定芽塊增殖之影響........................................................................27五) 不定芽、擬胚以及胚性癒合組織之切片解剖觀察...........................................27論 (Discussion) ........................................................................................................71、誘導石蒜雙核型雜交選系(26-355)鱗片誘導逆分化及再生.............................71一) MS培養基組合生長調節劑之處理.....................................................................71二)試驗基本鹽類組合生長調節劑之處理.................................................................72三)光照與黑暗處理.....................................................................................................72、石蒜雜交選系細胞懸浮系統之條件...................................................................73一)基本鹽類以及有機物添加.....................................................................................74二)石蒜懸浮細胞生長發育之影響因子.....................................................................75三)胞外pH調控對於細胞生長發育之影響..............................................................75.胞外pH變化與細胞生長發育之影響......................................................................76.添加pH緩衝物質對於胞外pH值與細胞生長速率之影響...................................77、平板培養................................................................................................................77一)添加生長調節劑種類對於平板培養之影響.........................................................77二)細胞團大小對於懸浮細胞再生之影響.................................................................78、短暫浸漬系統對於不定芽塊增生之影響............................................................78考文獻........................................................................................................................8