- 學位論文
石蒜雙核型雜種幼花序培養再生作用及小植株建立
Regeneration and Plantlet Establishment from Young Floret Culture in Dikaryotype Hybrids of Spider Lily (Lycoris spp.)
摘要
石蒜 ( Lycoris spp. ) 為相當具發展潛力之夏季球根花卉，幼年期長達5至6年且成年母球自然分球效率僅1.8倍，因此必須建立微體繁殖系統以供新雜交選系之推展。本試驗參試種間及所育成MT-A雜種選系，探討不犧牲母球之加速繁殖技術、建立石蒜幼小花起始之再生系統，比較種間及組織部位再生能力差異性、影響不定芽發生因子、重複增生技術及評估繁殖效率，進而縮短育種年限。 幼花培養，需以生長素組合BA 10 mg/L以誘導出大量不定芽。參試幼小花不同部位組織接種於含NAA 10 mg/L及BA 10 mg/L培養基，以花梗較具再生活力，其次為花被，子房較差，舉換錦石蒜(L. sprengeri)雜交金花石蒜(L. aurea)之選系為例，不定芽再生頻率分別為83.3%、47.6%及22.2%。再生途徑方面，花梗及子房培殖體可誘導直接不定芽發生，花被則為間接不定芽發生，再生時間延緩，但其培殖體數目較多。金花石蒜與不同A核型種雜交組合選系，以帶有紅藍石蒜(L. haywardii)及換錦石蒜基因組者較具再生能力，花被培殖體再生不定芽頻率介於40-82.8%，而帶有紅花石蒜(L. radiata)基因組，再生能力普遍較低，介於8.1-46.2%。 花序衍生不定芽長成小鱗莖，加以十字分切誘導不定芽及重複繼代增生培養，將不定芽叢分切培養於NAA 2 mg/L及BA 10 mg/L之培養基中效果較好，經3個繼代週期後，總繁殖倍率可達108.5倍。體外結球及根系誘導試驗，將芽體移至液體培養基中添加蔗糖濃度90 g/L，有助小鱗莖肥大生長，2個月後小鱗莖直徑可由3 mm增大為9.3 mm。於蔗糖濃度60 g/L，液態培養基中添加KH2PO4 340 mg/L可促使蔗糖有效利用，直徑為8.6 mm，葉長可達9 cm。單獨使用NAA及IAA兩生長素對石蒜小鱗莖生長發育無顯著效應，組合NAA 1 mg/L及 BA 2 mg/L，可明顯提升葉片數至2.4枚，葉長達8.7 cm，但BA濃度大於1 mg/L根系生長被抑制。18℃下培養，鱗莖較小但可得較佳之葉數3.1枚、葉長9.3 cm及根數3.3。大小不同之小鱗莖移植出瓶，小鱗莖周徑大於2.4 cm，存活率可達100%，周徑小於2.4 cm，存活率下降至81.3%。 石蒜鱗片以2,4-D 1 mg/L及BA 5 mg/L培養8週後，可於鱗片基部產生大量癒合組織，並可產生叢生之體胚，每個培殖體平均體胚數為3.2個。以胚性癒合組織做為細胞培養之起始培殖體，空胞數量較非胚性癒合組織處理少，但尚未能培養出均質之細胞系。
並列摘要
The spider lily (Lycoris spp.) possesses hysteranthous and graceful flowering potential to develop as novel summer bulb plants. The juvenile period of Lycoris spp. requires 5-6 years and the natural multiplication rate is limited 1.8 times after adult stage. Thus, it is necessary to establish rapid micropropagation system benefit for the extension of new selection. The pedicle, ovary and perianth explants were inoculated on MS medium supplemented with NAA 10 mg/L and BA 10 mg/L. The pedicle explants produced more buds than perianth or ovary. For example, the regeneration rate of adventitious bud in pedicle, perianth and ovary on selection of L. sprengeri x L. aurea were 83.3%, 47.6% and 22.2%. Direct adventitious bud formation was induced from pedicle and ovary, and indirect caulogenesis take place in perianth explant. The regeneration capacity in MT-A hybrids 2n=18(4M+3T+11A) of L. aurea 2n=14 (8M+6T) crossed with different A karyotype species 2n=22 (22A), which display higher multiplication rate between 40-82.8% in L. haywardii or L. sprengeri than 8.1-46.2% in L. radiata genome containing hybrids. Adventitious bud originated from inflorescence grow into bulb, then it was cross cutted and cultured on the medium to induce adventitious buds. More adventitious buds formed when subcultured on the medium supplemented with NAA 2 mg/L and BA 10 mg/L. After 3rd subculture period, total regeneration rate was 108.5 times. In vitro bulbing and rooting, buds were transferred to liquid medium supplemented with sucrose 90 g/L to promote bulb growth. The diameter of bulb became 9.33 mm after culture for 2 months. Supplemented with KH2PO4 340 mg/L in the liquid medium can promote the utilizing of sucrose by bulblet. The diameter and leaf length of bulb were 8.6 mm and 9 cm. There is no significant influence on bulb growth cultured in the medium supplemented with NAA or IAA. The leaf number and leaf length of bulb were reached 2.4 and 8.7 cm when cultured in the liquid medium supplemented with NAA 1 mg/L and BA 2 mg/L. Root growth was limited when the concentration of BA higher than 1 mg/L. Bulb cultured in the 18℃ chamber has better result, the leaf number, leaf length and root number were 3.1, 9.3 cm and 3.3. Bulbs with different size were transplanted to growth chamber. The survival rate was 100% when the perimeter of bulb bigger than 2.4 cm. If perimeter below 2.4 cm the survival rate only 81.3%. Medium supplemented with 2, 4-D 1 mg/L and BA 5 mg/L was suitable for embryo production. 3.2 embryos were obtained from each explant after culture for two months. The cells releasing from embryogenic callus is better than non-embryogenic callus, but still could not form more uniform cell line after 4 months.