探討蝴蝶蘭葉培植體直接體胚發生之影響因子

Abstract

Phalaenopsis amabilis 及 Phalaenopsis Nebula 約 1 cm 的幼葉培植體，置於含 3 mg/l TDZ 之1/2MS培養基，黑暗培養一個月後，不經癒傷組織即可直接於葉培植體的基部切口、葉片邊緣、葉尖或葉培植體的向軸面形成體胚 (somatic embryos)。葉培植體培養45天時，置換培養基將可提高每個培植體之平均體胚數，培養60天後，可形成多量之體胚。含 0.5 mg/l BA 培養基可顯著提高兩個月大的體胚存活率，且可獲得生長成正常之小植株。本結果顯示，由於蝴蝶蘭葉片可直接產生體胚，之後形成具形態雙極性的小植株，此植株再生系統應可應用於未來進行之基因轉殖等研究。 蝴蝶蘭 (Phalaenopsis amabilis 及 Phalaenopsis Nebula) 幼葉培植體，置於含 3 mg/l TDZ 之1/2MS培養基，其向軸面及背軸面皆會產生體胚，但仍以向軸面朝上擺放之體胚發生率較朝下擺放者高。光會抑制葉培植體形成體胚，且使葉培植體褐化比率提高。因此，葉培植體於黑暗下以向軸面朝上方式擺放較有利於直接體胚之發生。 Phalaenopsis amabilis 及 Phalaenopsis Nebula 之 1 cm葉培植體培養於五種 cutokinin (2iP, kinetin, BA, TDZ 及 zeatin) 的1/2MS培養基，皆促進葉培植體之體胚發生，而體胚主要發生於葉基切口及葉向軸面，若葉培植體未添加植物生長調節劑，將無體胚形成。Phalaenopsis amabilis 最好的處理是含 3 mg/l BA 的培養基，為80%，3 mg/l TDZ 次之，為65%。然而， 3 mg/l TDZ 之體胚數較高。Phalaenopsis Nebula在 3 mg/l TDZ 的體胚發生率最高，為65%。此外，20 μM ACC 有促進 Phalaenopsis amabilis 葉培植體體胚發生的效果，體胚發生率為35%。外施 auxin、GA3、ancymidol、polyamines、AgNO3 及 CoCl2 在體胚誘導上並無促進效果。 蝴蝶蘭 (Phalaenopsis amabilis) 幼葉培植體的體胚發生率及體胚數目受到培養基成份的影響。葉培植體較適於培養在1/2 MS 之基礎培養基。不含蔗糖的培養基無體胚形成且培植體易褐化。20 g/l 蔗糖就可滿足體胚發生之需求。培養基添加 170 mg/l NaH2PO4 及 0.25 g/l PVP 有利於葉培植體之體胚發生，活性碳則抑制體胚之形成。

Explants taken from 1 cm young leaves of Phalaenopsis amabilis and Phalaenopsis Nebula formed somatic embryos directly from wound surfaces, leaf tips, adaxial sides and edges of leaf explants without an intervening callus after one month of culture on 1/2 MS medium supplemented with 3 mg/l TDZ in darkness. Average of number somatic embryos per explant was promoted when subculture in 45 days of culture and produced large number of somatic embryos after 60 days of culture. The results show that enhanced the percentage of somatic embryos survival and develop into plantlets from two-month old embryos when cultured on a medium supplemented with 0.5-1 mg/l BA. The directly somatic embryogenesis of leaf explants in Phalaenopsis is successful for plant regeneration and this protocol could be useful for further genetic transformation in this orchid. Young leaf explants of Phalaenopsis amabilis and Phalaenopsis Nebula directly formed somatic embryos from both side of leaf explants. In addition, explants placed adaxial-side-up orientation on culture medium had significant higher percentage of embryogenesis than the abaxial-side-up orientation. Light regime inhibited direct embryo formation from leaf explants, and had higher percentage of browning than in darkness. In conclusion, adaxial-side-up orientation and culturing under darkness were more suitable for embryo induction from Phalaenopsis leaf explants. Explants taken from 1 cm leaves of Phalaenopsis amabilis and Phalaenopsis Nebula promoted the direct somatic embryogenesis from the wound surfaces and adaxial side of leaf explants on 1/2 MS medium supplemented with all five cytokinins (2iP, kinetin, BA, TDZ and zeatin). There was no any embryos formed on a hormone-free medium for leaf explants. The best response of Phalaenopsis amabilis was 80% and 65% of embryogenesis on medium supplemented with 3 mg/l BA and 3 mg/l TDZ respectively. However, 3 mg/l TDZ became the embryo higher in numbers. The highest percentage of embryogenesis in Phalaenopsis Nebula was 65% with 3 mg/l TDZ. In addition, the percentage of embryo formation was 35% on medium supplemented with 20 μM ACC. The presence of auxin, GA3, ancymidol, polyamines, AgNO3 and CoCl2 had no inductive effect on embryo formation. Both of the percentage of embryogenesis explants and the number of embryos per explant from young leaf explants of Phalaenopsis amabilis were affected by medium composition. Leaf explants was more suitable on 1/2 MS basal medium. There was no embryogenic response and explants necrosed on medium without sucrose, and should be supplement 20 g/l sucrose for the requirement on embryogenesis. The presence of 170 mg/l NaH2PO4 and 0.25 g/l PVP promoted the somatic embryogenesis on leaf explants. Besides, active charcoal inhibited the embryo formation on leaf explants.