- 學位論文
芭菲爾鞋蘭、文心蘭、軛瓣蘭及堇蜘蘭之微體繁殖
Micropropagations of Paphiopedilum, Oncidium, Zygopetalum and Miltassia
摘要
1. 芭菲爾鞋蘭 (Paphiopedilum ‘Alma Gaveart’) 由種子誘導之癒傷組織，培養於含5 mg/l IAA、0.1-5 mg/l NAA、0.5 mg/l 2,4-D、1 mg/l TDZ、0.5-5 mg/l NAA組合0.1-1 mg/l TDZ或0.5-1 mg/l 2,4-D組合0.1-0.3 TDZ的1/2 MS培養基，90-180天後，形成類原球體，然後發育為小苗。其中，以0.5 mg/l NAA組合0.1 mg/l TDZ處理，於培養100天後，30% 癒傷組織形成類原球體，平均芽數為2.8。生長素促進芭菲爾鞋蘭癒傷組織增生、類原球體形成、芽體增殖及發根；細胞分裂素則導致癒傷組織褐化及芽體玻璃質化。再生小苗的根，培養於含1mg/l TDZ及5 mg/l 2,4-D的1/2 MS培養基，一個月後形成癒傷組織。 2. 文心蘭 (Oncidium Gower Ramsey及Onc. Sweet Sugar) 葉培殖體置於4.54 μM TDZ之1/2 MS培養基，黑暗下培養1個月後，形成多量體胚。添加10-30 g/l葡萄糖、10-30 g/l果糖、20-60 g/l蔗糖、1 μM水楊酸與4.54 μM TDZ、0.01-1 μM乙醯水楊酸與4.54 μM TDZ或20 μM ACC與4.54 μM TDZ，可提高Onc. Gower Ramsey及Onc. Sweet Sugar之體胚發生效率。水楊酸、乙醯水楊酸、AgNO3、CoCl2、GA3、ancymidol及動物性荷爾蒙抑制體胚發生。此外，1-5 μM ACC、10 μM spermidine或4.54 μM TDZ組合5-10多元胺，可促進Onc. Gower Ramsey的體胚發生，而4.54 μM TDZ組合0.1 μM progesterone之處理，則促進Onc. Sweet Sugar之體胚發生。 3. 軛瓣蘭 (Zygopetalum mackayi) 之莖節置培植體於含0-1 mg/l TDZ的1/2 MS培養基，光照下培1個月後，形成類原球體及叢生芽，每一培植體在1 mg/l TDZ培養基培養2個月後形成17芽。長期繼代於0.3-1 mg/l TDZ抑制抽芽。因此，宜採用兩段式培養，以0.3-1 mg/l TDZ誘導類原球體和芽體之形成及增殖，然後移至不含生長調節劑或含0.01 mg/l GA3的1/2 MS培養基，以誘導抽芽及發根。 4. 堇蜘蘭 (Miltassia Olmec ‘Kanno’) 之幼嫩花苞，在黑暗中培養於含3 mg/l TDZ及3 mg/l 2,4-D，或1-3 mg/l TDZ及3-10 mg/l NAA的1/2 MS培養基2個月後，於花被切口及鄰近切口處，或子房壁鄰近切口處形成不定芽。此等不定芽可在原培養基中繼代及增殖。其中，以1 mg/l TDZ組合10 mg/l NAA及3 mg/l TDZ組合3 mg/l NAA之處理誘導的芽數最高，經過2個月的芽體誘導及3個月的芽體增殖培養後，各得18及17芽。
並列摘要
1. Seed-derived callus of Paphiopedilum ‘Alma Gaveart’ was used to test the effects of auxins and cytokinins on PLB regeneration and subsequent plant formation. Plantlets were obtained via PLB formation after 90-180 days when callus was cultured on 1/2 MS media supplemented with 5 mg/l IAA, 0.1-5 mg/l NAA, 0.5 mg/l 2,4-D, 1 mg/l TDZ, 0.5-5 mg/l NAA and 0.1-1 mg/l TDZ or 0.5-1 mg/l 2,4-D and 0.1-0.3 TDZ. The best response was observed at 0.5 mg/l NAA combined with 0.1 mg/l TDZ, 30% callus formed PLB and 2.8 shoots were obtained. In general, auxins enhanced callus proliferation, PLB formation, shoot multiplication and rooting. By contrast, cytokinins cause browning of callus and vitrification of shoots. In addition, root segments taken from callus-derived plantlets were used to induce callus formation. After one month of culture, yellowish callus formed from root tips and wounded regions of root explants on 1/2 MS medium supplemented with 1 mg/l TDZ and 5 mg/l 2,4-D. 2. Large number of somatic embryos directly formed from leaf explants of Oncidium Gower Ramsey and Onc. Sweet Sugar after one month of culture on 1/2 MS medium supplemented with 4.54 μM TDZ in darkness. Supplementation of 10-30 g/l glucose, 10-30 g/l fructose, 20-60 g/l sucrose, 1 μM salicylic acid and 4.54 μM TDZ, 0.01-1 μM acetylsalicylic acid and 4.54 μM TDZ or 20 μM ACC and 4.54 μM TDZ to culture media promoted embryogenesis efficiency of Onc. Gower Ramsey and Onc. Sweet Sugar. Salicylic acid, acetylsalicylic acid, AgNO3, CoCl2, GA3, ancymidol and animal sex hormones inhibited somatic embryogenesis. In addition, 1-5 μM ACC, 10 μM spermidine, 4.54 μM TDZ combined with 5-10 μM polyamines promoted embryogenesis of Onc. Gower Ramsey while 4.54 μM TDZ combined with 0.1 μM progesterone promoted embryogenesis of Onc. Sweet Sugar. 3. Protocorm like bodies (PLBs) and multiple shoots were obtained from nodal explants of Zygopetalum mackayi after one month of culture on 1/2 MS medium supplemented with 0-1 mg/l TDZ under light. Seventeen shoots were obtained from one explant after two months of culure on a medium supplemented with 1 mg/l TDZ. Shoot elongation was inhibited by 0.3-1 mg/l TDZ after long-term subculture. Therefore, two-step culture for propagating this orchid was established. Fristly, nodal explant were placed on 1/2 MS medium supplemented with 0.3-1 mg/l TDZ to induce formation and multiplication of PLBs and shoots. Secondly, the clusters of PLBs and shoots were transferred onto media devoid of plant growth regulators or supplemented with 0.01 mg/l GA3 for further shoot elnogation and root formation. 4. Explants taken from flower buds of Miltassia Olmec ‘Kanno’ formed adventitious shoots from the wounded tissue of parianthes and ovary walls after 2 months of culture on 1/2 MS media supplemented with 3 mg/l TDZ and 3 mg/l 2,4-D, or supplemented with 1-3 mg/l TDZ and 3-10 mg/l NAA in darkness. The adventitious shoots could be subcultured and muliplicated on the same media under light condition. After two months of culture for shoot induction and three months of culture for shoot multiplication, 18 and 17 shoots were obtained from one single explant in treatments of 1 mg/l TDZ combined with 10 mg/l NAA and 3 mg/l TDZ combined with 3 mg/l NAA, respectably.