無患子體胚誘導之研究

Abstract

本研究採集台大校園無患子（Sapindus mukorossi Gaerm）之未成熟胚為材料，藉由組織培養進行癒合組織誘導並探討不同條件對體胚發生的影響。並期望能利用懸浮細胞培養技術及體胚分化培養技術，建立經濟、穩定、有效之體胚分化系統，作為品種篩選後無患子種苗大量繁殖之參考依據。 將無患子未成熟胚培養於WPM培養基添加0.1mg/L NAA可以促進無患子發根及發芽，將已發育之下胚軸切下繼代培養於WPM培養基添加0.3 mg/L Kinetin，4週後可誘得癒合組織。於本試驗中，無患子體胚誘導的最適條件為切取約0.5㎝×0.5㎝×0.5㎝的癒合組織團塊繼代培養於WPM基礎培養基含2%蔗糖，修正2倍NH4+含量中，並且添加0.1 mg/L NAA和0.05mg/L TDZ，照光強度平均為28μEm-2s-1，光週期為16小時的條件下培養8週後，可得到最佳的體胚誘導率。體胚發生後若要大量繁殖繼續誘導二次體胚，可於培養基中添加0.1∼0.5 mg/L的GA3，可以促進體胚生成二次體胚。反之若是要使體胚成熟化發育成苗，可以增加培養基中GA3的濃度至2 mg/L，則可促進無患子的體胚成熟化，提高無患子體胚的胚苗轉換率。 在細胞懸浮培養方面，以無患子下胚軸產生之癒合組織為懸浮培養材料，經2 ~ 4個月的繼代培養後，逐漸有細胞團和細胞質濃密的細胞出現。因此無患子可能需要更長時間的繼代培養，才能得到游離的胚性癒合組織。

Immature embryos of soap berry (Sapindus mukorossi Gaerm.) in the National Taiwan University campus were collected for callus induction via tissue culture techniques to study on the effects of different factors on somatic embryogenesis. Techniques of cell suspension culture and embryogenesis were employed to establish an economic, stable and effective system for micropropagation of seedling and to facilitate selection and breeding of superior strains of soap berry. While immature soap berry embryos being cultured in WPM media, addition of 0.1mg/L NAA could promote rooting and budding. Callus could be induced after 4 weeks by cutted grown hypocotyls sub-cultured on WPM medium with 0.3 mg/L kinetin. In this work, the optimal cultivation condition for somatic embryogenesis induction of S. mukorossi was made by sub-culturing 0.5 cm × 0.5 cm × 0.5 cm callus clump on WPM medium with 2% sucrose, double NH4+content, 0.1 mg/L NAA and 0.05mg/L TDZ, under 28μEm-2s-1 average light intensity, 16 hrs photoperiod for 8 weeks. Then, optimal somatic embryogenesis induction ratio could be achieved. In order to induce secondary somatic embryo after embryogenesis, 0.1 ~ 0.5 mg/L GA3 could be added in medium to promote secondary somatic embryogenesis. To promote seedling, concentration of GA3 could be increased to 2 mg/L in medium to promote the embryo maturation and the ratio of emblings conversion. In the aspect of cell suspension culture, using callus induced from soap nut hypocotyls of as starting materials, some cell microsphere and cell with dense cytoplasm gradually appeared after sub-culturing for 2 ~ 4 months. Hence, longer sub-culturing time could be required to obtain extricated embryogenic callus.