臺灣一葉蘭‘梅雪’與‘楓星’之微體繁殖

Abstract

臺灣一葉蘭(Pleione formosana Hayata)是臺灣原生的落葉性蘭花，球莖運輸便利極具外銷日本歐美市場潛力，現只臺大山地實驗農場擁有品種權，但雖有優良之商業品種，受限於繁殖倍率低而無法大量供應外銷及內銷市場。為快速大量生產臺灣一葉蘭商業品種並維持品種特性，本研究採新品種‘梅雪’與‘楓星’，取冷藏過的球莖芽體，以組織培養誘導體胚或不定芽，其後探討不同培養基及光源條件對其增殖生長的影響，調整培養基中生長調節劑之種類與濃度，以提高次級培植體繼代後的增殖率，並調查不同培養光源與光度對組培苗生長之影響。最後調整組培苗出馴化期間的光源與光度，期望能提高移植後的存活率與生長量，並探討臺灣一葉蘭組培苗的出瓶適期。 臺灣一葉蘭‘梅雪’球莖於0、2、4、6、8、10、12、15℃黑暗生長箱中冷藏4、6、8週後，取第四芽培養於添加20 g•L-1 sucrose、4 g•L-1 gelrite、0.15 mg∙L-1 NAA、0.2 mg∙L-1 BA之1/2 Murashige & Skoog (MS)培養基，以2-8℃冷藏6週者在培養24週後有較高的增殖率。取5℃冷藏6週的‘梅雪’球莖頂芽、第四芽及第五芽為培植體，僅第四芽與第五芽培養於含20 g•L-1 sucrose、3 g•L-1 Tryptone、4 g•L-1 gelrite及2 g•L-1活性碳的1/2 MS培養基後可增殖，並添加1.2、2.4 mg•L-1 Dicamba或1.1、2.2 mg•L-1 Picloram組合0.2 mg•L-1 BA可誘導直接體胚發生。 將臺灣一葉蘭‘梅雪’及‘楓星’第四芽培養再生之叢生芽，分切培養於含不同濃度NAA及BA之1/2 MS中，培養8週後以0.2 mg•L-1 NAA組合0.4 mg•L-1 BA處理誘導的體胚數最多，誘導出的體胚與無菌播種的臺灣一葉蘭小苗有相似的發育過程，體胚繼代至含0、0.2、0.4 mg•L-1 NAA組合 BA的1/2 MS培養基，可從初級體胚表面產生次級體胚，次級體胚與初級體胚有相似的發育過程，NAA濃度越高越能促進體胚發育成小苗，體胚發育成的小苗經埋蠟切片可觀察到獨立封閉的維管束構造。取臺灣一葉蘭‘梅雪’與‘楓星’組培苗分切為葉尖、葉基及球莖薄片進行培養，僅帶有芽體的球莖薄片可增殖，適用的培養基為添加0.2 mg∙L-1 2, 4-D或NAA 組合0.2 mg∙L-1 BA之1/2 MS培養基。 培養於LED白光(19% 紅 + 52% 綠 + 29% 藍)下的組培苗，較LED紅光、LED藍光、LED紅藍混合光及螢光燈有高的芽數、鮮重、球莖寬度與葉長。適合臺灣一葉蘭組培苗的培養光度為30 μmol•m-2•s-1。組培苗出瓶後在光度30、60及90 μmol•m-2•s-1下馴化兩週期間，雖然30 μmol•m-2•s-1處理的Fv/Fm值及淨光合作用速率較高，但移植後60及90 μmol•m-2•s-1處理有較高的存活率及生長量。組培苗出瓶後於60 μmol∙m-2∙s-1不同光質下馴化3週，以LED白光(19% red + 52% green + 29% blue)處理者在馴化期間有較高的淨光合作用速率值，且移植後的存活率和生長量亦較高。將瓶內培養8、12、16週已結球並帶根的組培苗出瓶，之後於光度60 μmol•m-2•s-1下馴化3週並移植，以培養12、16週者有較高的移植後存活率、鮮重、球莖寬度與根數。

Pleione formosana Hayata is a Taiwan-native deciduous orchid species suitable for transportation to meet European and Japanese market`s demand. Though cultivars have been developed but sales were limited due to low propagation efficiency. This study aimed to determine suitable conditions for mass propagation of P. formosana ‘Mei Snow’ and ‘Feng Star’ by somatic embryogenesis or multiple shooting. Bud from cold stored P. formosana corms were taken as explants, and cultured on mediums containing various plant growth regulator combinations. Light intensity and quality during in vitro and ex vitro acclimatization were tested to increase growth and ex vitro survival. The effect of in vitro cultured duration on ex vitro growth was also studied. The fourth bud on P. formosana corms after cold storage at 0, 2, 4, 6, 8, 10, 12, and 15oC for 4, 6, and 8 weeks were taken as explants. The fourth bud explants were cultured on 1/2 Murashige & Skoog (MS) medium containing 20 g•L-1 sucrose, 4 g•L-1 gelrite, 0.15 mg∙L-1 NAA (naphthalene acetic acid), and 0.2 mg∙L-1 BA (benzyl adenine purine). Those stored at 2-8oC for 6 weeks showed higher multiplication rate when cultured after 24 weeks. Apical bud, the fourth bud, and the fifth bud of P. formosana ‘Mei Snow’ stored at 5oC for 6 weeks were taken as explants. Only the fourth bud and the fifth bud explants were able to regenerate after cultured on 1/2 MS medium containing 20 g•L-1 sucrose, 3 g•L-1 Tryptone, 4 g•L-1 gelrite, and 2 g•L-1 activated charcoal. Direct somatic embryo were induced when the fourth and the fifth bud explants were cultured on 1/2 MS medium supplemented with 1.2, 2.4 mg•L-1 Dicamba or 1.1, 2.2 mg•L-1 Picloram and 0.2 mg•L-1 BA. Multiple shooting regenerated from the fourth bud culture were divided as secondary explants, and cultured for 8 weeks on 1/2 MS medium supplemented with NAA and BA combinations. Results showed those cultured on 0.2 mg•L-1 NAA combined 0.4 mg•L-1 BA induced more somatic embryos, with growth phase similar to seed-develop plantlets. Secondary somatic embryos were induced from surface of primary somatic embryos after subcultured to 1/2 MS medium containing 0, 0.2, and 0.4 mg•L-1 NAA and BA combinations. Secondary somatic embryos shared similar growth stage with primary somatic embryos. Somatic embryos tended to develop plantlets with increasing NAA concentration. Anatomical reservation showed that plantlets developed from somatic embryo had independent isolated vascular bundle. Leaf tip, leaf base, and corm slice from in vitro plantlets were also taken as explants, but only corm slices with buds could regenerate to plantlets. More somatic embryos were induced from those cultured on 1/2 MS medium supplemented with 0.2 mg∙L-1 2, 4-D or NAA combined with 0.2 mg∙L-1 BA. Plantlets cultured with LED (light emitting diode) white light (19% red + 52% green + 29% blue) had more buds, fresh weight, corm diameter, and leaf length than those cultured with LED red, LED blue, LED red plus blue light, and fluorescent lamp. Suitable light intensity for in vitro culture was 30 μmol•m-2•s-1 PPF. Ex vitro plantlets were acclimatized under 30, 60, and 90 μmol•m-2•s-1 PPF for 2 weeks. Results showed that plantlet acclimatized under 30 μmol•m-2•s-1 showed higher Fv/Fm value and net photosynthesis, but those acclimatized under 60 and 90 μmol•m-2•s-1 had higher survival rate and better growth after transplanting. En vitro plantlets were also acclimatized under various light quality at 60 μmol•m-2•s-1 for 3 weeks. Results showed that plantlets under LED white light had higher net photosynthesis, survival percentage, and more growth after transplanting. Plantlets cultured in vitro after 8, 12, and 16 weeks were transplanted and acclimatized under 60 μmol•m-2•s-1 for 3 weeks. Results showed that higher survival rate, fresh weight, corm diameter, and root number were recorded in plantlets cultured for 12 and 16 weeks then that cultured for 8 weeks.