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Microprogation of Hippeastrum via Twin Scaling
本研究以孤挺花(Hippeastrum hybridum Hort)三年生鱗莖(周徑20-26cm)為材料，探討瓶內培養時不同消毒方式、培養條件對雙鱗片微體繁殖之影響，以及移植瓶外不同介質成分及穴盤型式大小對小鱗莖生長之影響。利用2% Clorox處理''Apple Blossom'' 20分鐘後，即可降低污染率至0%，而''Best Seller''及''Red Lion''必須以3% Clorox處理30分鐘後，才可降低污染率至30%以下；Clorox消毒時間以20分鐘為宜，消毒40分鐘後污染率為0%，但培植體幾乎不生長。不同成熟度雙鱗片之鱗莖再生不論在內層或外層均無顯著差異，而葉片長度以中間層最長。
孤挺花''Red Lion''經由組織培養之小鱗莖，再四分切培養於不同鹽類濃度中，以全量及半量MS最有利鱗莖生長，當移出瓶外種植時，結果顯示1/4MS培養之鱗莖適應能力最好，具有最高相對生長率。於不同蔗糖濃度試驗中，四分切培養於30-40g/l蔗糖培養基中生長情形最好，但移出瓶外種植時以由60-80g/l蔗糖中培養者具有最高相對生長率，另外培養基中添加活性碳有利根生長，降低根褐化率。四分切鱗片放置方式，對''Apple Blossom''不論向軸面、背軸面或直立式，小鱗莖再生並無顯著差異；而''Red Lion''以背軸面培養方式之再生小鱗莖數最多，但誘導癒合組織形成上以向軸面最好。
孤挺花''Red Lion''四分切培養於含有2mg/l BA培養基中，有較多小鱗莖形成；至若於2mg/l BA外加0.75mg/l 2,4-D之培養基中進行培養，再移至不含生長素之培養基中，則可得到最高體胚形成率，若於含kinetin 0.25-2mg/l者移至不含生長素培養基中，明顯較不含kinetin對照組得到更高體胚形成率及癒合組織；體胚之芽體形成以培養在0.1或1mg/l BA液體培養基中較好。而TDZ試驗，小鱗莖再生率並沒有顯著性差異，只有根數會隨著TDZ濃度增加而降低。發根試驗以NAA 0.5-1.5mg/l處理的結果比對照組具有較多根數。
小鱗莖以完整植株生長勢最好，但輕微受傷並不會影響其生長；以泥炭土作為栽培介質，小鱗莖生長最好；而小鱗莖生長會隨著穴格增大而提高相對生長率，尤其以方-1 (380cm3)及方-2 (120cm3)的表現最佳，而在二次根生長以方-1及圓-1 (150cm3)再生數目多，整體而言較大穴格對鱗莖生長是有利的。但如果考量可種植數量及經濟因素，則以方-2較適合作為大量鱗莖種植時使用。
To understand the effect of different disinfection methods and cultural conditions on double scaling during in vitro culture and the effect of culture medium and plug type on bulblet growth in vivo were of great interest. Three-year-old Hippeastrum hybridum Hort. stock bulbs with 20-26cm diameter were used in this study. By using 2%, 3% Clorox surface disinfection treatment on ''Apple Blossom'' and ''Best Seller'' and ''Red Lion'' for 20 and 30 minutes could reduce the contamination rate to 0% and under 30%, respectively. The proper disinfection time is the best under 20 minutes. Although 40 minutes minimized contamination rate to 0%, bulblets could barely grow. When double scaling of different maturity bulb scale, no significant has found on inside or outside section. However, leaf length is the longest on middle section.
While culture ''Red Lion'' quarter chips of the bulblet (QCB) cultured under different salt strength, both of full and half MS media is the best for bulblet growth in vitro. Relative growth of bulblet grew best under 1/4 MS medium when they were transplanted ex vitro. Under different sucrose medium concentration, 20 - 40 g/l grew best in vitro, 60 - 80 g/l has the highest relative growth rate when transplant ex vitro. Necrosis root reduced when activated charcoal was added. Regarding the propagation placing position, ''Apple Blossom'' produced large amount of bulblets whenever the position was. However, ''Red Lion'' produce largest amount of bulblets on backside down and induced largest amount callus while inside down.
In the effect of plant growth regulator on ''Red Lion'' bulblet proliferation, 2 mg/l BA medium formed the largest amount of bulblets. Cultured QCB on 2 mg/l BA + 0.75 mg/l 2,4-D and transplanted to no plant growth regulator (PGR) media obtained the highest amount of somatic embryos. Subcultured QCB from kinetin 0.25 - 2 mg/l to no PGR media could gain more somatic embryos and calli than that of CK. Liquid media contained 0.1 or 1 mg/l BA resulted good in somatic embryogenesis formation. Too high BA in media concentration will enable explants to development organgenesis. No bulblet regeneration has found on TDZ treatment, however, root number reduced as TDZ concentration was increased. NAA 0.5 - 1.5 mg/l has higher root number result than that of CK.
Whole healthy bulblets grew best, a little injury did not affect bulblet growth, and peatmoss is the best ex vitro growing media for bulblets cultivation. Relative growth rate of bulblet increased as plug cell size was increased, especially on square-1 (380cm3) and square-2 (120cm3). Secondary rooting number was best on square-1 and round-1 (150cm3). Generally, larger cell (over 120cm3) was suitable for bulblet growing ex vitro. However, considering planting amount and efficiency, square-2 was the best.
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