石蒜屬雙核型雜種花序體外培養及多倍體誘導

Abstract

金花石蒜花型優美且切花品質佳，屬極具發展潛力之新興夏季球根花卉，且同時具有多種藥用鹼可供醫學方面應用，但石蒜幼年期長及自然分球效率低，為新品種育成與推廣栽培之極大限制主因，因此本論文除探討如何不犧牲母株進行繁殖外，並進一步利用不同培養方式針對以金花石蒜為親本之雜交選系與雙核型雜種回交至金花石蒜之後裔(試交代)，進行加速大量繁殖。 本試驗參試材料為本實驗室育成之金花石蒜雙核型雜交種及其試交代，選系包含紅花石蒜×換錦石蒜、金花石蒜×紅花石蒜與金花石蒜×換錦石蒜。參試品系選用幼花序進行培養，均以幼花被培植體再生率最高，而生長調節劑組合試驗各品系再生誘導效果不同，但以2,4-D組合BA之處理誘導不定芽再生比例較高可達100%，而2 mg/L NAA組合10 mg/L BA處理於部分品系不定芽誘導效果也可達70%以上。雙核型雜種幼花被衍生芽塊經繼代培養8週後，增殖倍率均可達1倍以上，約可再生40至80個不定芽。 以再生之小鱗莖為二次培植體，進行十字分切後固體培養8週後，A核型群種間雜種以0.5 mg/L NAA組合5 mg/L BA之處理，平均每個小鱗莖可再生10.36個芽，MT-A核型種在0.5mg/L 2,4-D組合5 mg/L BA可再生9.08個芽；三種間雜種則以0.5 mg/L NAA組合5 mg/L BA可再13.88個芽最多。另以短暫浸漬系統進行鱗莖分切培養，A核型種內雜種(LRM8709 × LS)與MT-A雙核型種(LA × LRK2)於各試驗處理再生不定芽數均較固體培養多，其中A核型種以0.5 mg/L 2,4-D組合5 mg/L BA可再生108個芽，增殖倍率可達21.6倍。而MT-A核型種則以0.5 mg/L NAA組合5 mg/L 2-ip再生76個不定芽最多，其增殖倍率為16.89。 石蒜A核型種間雜種鱗莖直徑為1至2 mm之芽置於蔗糖濃度為90 g/L之液體培養基內，培養8週後鱗莖直徑可達10.2 mm，而MT-A雙核型雜種品系小鱗莖直徑則可至10.75 mm。將鱗莖直徑5至10 mm小鱗莖進行發根試驗，以培養基內有添加活性碳之處理，發根情形良好，且移植出瓶後存活率可達90%以上，出瓶6週後葉數達3至4枚，相當於實生苗栽培2到3年可達之葉片數。 針對MT-A雙核型雜種低落之稔實性，擬以人為方式進行多倍體誘導，以鱗莖鱗片培植體進行誘變試驗後，再生不定芽並進行單株培養取其根尖進行染色體倍數檢定，品系LA × LRK4經二次根尖染色體檢定之擬四倍體株，以流式細胞儀進行DNA含量檢定，除100 μM Oryzalin 72hr pH 5.7 -3單株為鑲崁體外，其餘各植株地上部均已回復為二倍體性狀。

Lycoris aurea Taiwanese native flower has been grown for cut flower trade. Lycoris species also have high economic value for their medicinal alkaloids. They require four to five cycles from seed to flower, and the propagation rate is restrained to nature. The purpose of this research was not only to develop a method for propagation of dikaryotype MT-A hybrids and testprogenies of Lycoris spp, but also to use different culture container for rapid mass propagation. Experimental materials were dikaryotype MT-A hybrids and testprogenies of Lycoris spp. including of L.radiata × L.sprengeri, 2n = 22 (22A), L.aurea × L.radiata, 2n = 18 (4M+3T+11A) and L.aurea × L.sprengeri, 2n = 18 (4M+3T+11A). Immature inflorescenes segments were used for callus and adventitious buds initiation. The immature perianth produced the highest regeneration rate, and the adventitious bud inducing rates in the medium with 2 mg/L 2,4-D and 5 or 10 mg/L BA were more than other treatments. The medium with 2 mg/L NAA and 10 mg/L BA also showed good regeneration effect in some lines. After 8 weeks of subculture, the immature perianth-induced buds regenerated 40 to 80 buds, and the multiple rate was 1. In vitro grown bulblets with 5 mm diameters were cut into four segments for adventitious buds initiation. After 8 weeks of solid culture, the A specific karyotype and trispecific origin dikaryotype hybrid inoculated in medium with 0.5 mg/L NAA and 5 mg/L BA regenerated 10.36 and 13.88 buds, and the MT-A dikaryotype hybrids inoculated in medium with 0.5 mg/L 2,4-D and 5 mg/L BA regenerated 9.08 buds. By using TIS as culture container for segments adventitious buds initiation, the A specific karyotype (LRM8709 × LS) and MT-A dikaryotype hybrids (LA × LRK2) had much more adventitious buds regeneration than solid culture in all treatments. And the greatest result showed in the medium with 0.5 mg/L 2,4-D and 5 mg/L BA which induced 108 buds, and the multiple rate was 21.6 of A specific karyotype. MT-A dikaryotype hybrids had the greatest adventitious buds regeneration in the medium with 0.5 mg/L 2,4-D and 5 mg/L 2-ip which induced 76 buds, and the multiple rate was 16.89. The different karyotype buds with1 to 2 mm diameters in the medium with 90g/L sucrose after 8 weeks, the buds of A specific karyotype and MT-A dikaryotype grew 10.2 and 10.75 mm individually. After culturing in medium with activated charcoal , the bulblets formed root system. The survival rate of plantlets were more than 90%, and the leaf number grew to more than 3. For restoring the fertility of MT-A dikaryotype hybrids, the scale segments were used as explants for artificial polyploidy induction. The chromosome of root tips were tested after the adventitious buds formed plantlet. The regenerated leaves of pre-tetrapolyploidies were tested DNA content by flow cytometry. Besides the plantlet treated with 100 μM Oryzalin 72hr at pH 5.7 was chimera, the other were reverted to diploidy.