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研究生:梁智超
研究生(外文):Chih-Chao Liang
論文名稱:夏蓳種間雜交倍體數選拔及誘變育種
論文名稱(外文):Selection and Mutation Breeding of Interspecific Polyploid Torenia Hybrid
指導教授:葉德銘葉德銘引用關係
口試委員:朱建鏞沈榮壽陳福旗
口試日期:2014-07-09
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:園藝暨景觀學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:95
中文關鍵詞:疊氮化鈉體胚重瓣花雄蕊辦化
外文關鍵詞:Sodium AzideSomatic embryoDouble flowerStamen Petaloid
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摘要
夏蓳(Torenia fournieri Linden)為臺灣夏季重要花壇植物,株型直立不耐低溫,前人利用夏蓳‘Clown Rose’與臺灣原生毛葉蝴蝶草(T. benthamiana Hance)之種間雜交後代(Cb)具有耐低溫及植株生長匍匐性之特點,並經秋水仙素處理獲得Cb5-M612M1。本研究透過選拔Cb5-M612之自交後代(S1),分析其花朵性狀並建立不同培植體來源搭配疊氮化鈉之微體繁殖技術,期能大量繁殖並開發新品系。
以流式細胞儀檢測Cb及Cb5-M612 之葉片細胞DNA含量,結果顯示Cb5-M612為近四倍體。Cb5-M612經自交後觀察其後代花朵性狀,Cb5-M612S1及Cb5-M612S2側花瓣邊緣鋸齒狀較全緣為顯性,兩族群後代側花瓣全緣與鋸齒後代數量比符合1 : 15 (χ2 = 1.65 <χ20.05,2 = 5.99),Cb5-M612S1及Cb5-M612S2之唇瓣皆有黃點性狀。Cb5-M612S1花冠長寬出現分離情形,人為選拔大花單株Cb5-M612-S34,其小花梗長度、苞片長度及花冠面長度與夏蓳‘Clown Rose’無顯著差異。
以日/夜溫30/25與15/13 oC處理Cb5-M612S1與夏蓳‘Clown Rose’,並比較植株存活率、葉綠素計讀值、葉片相對傷害值與葉綠素螢光值。結果顯示低溫15/13 oC處理下,Cb5-M612S1皆能維持正常生長,然夏蓳‘Clown Rose’於15/13 oC之存活率僅50%。Cb5-M612S1於低溫15/13 oC仍保持較高之葉綠素計讀值。於30/25 oC及15/13 oC處理下,Cb5-M612S1之葉片細胞膜相對傷害值均較夏蓳‘Clown Rose’低。Cb5-M612S1於30/25與15/13 oC處理之Fv/Fm值無明顯差異,而夏蓳‘Clown Rose’於 15/13 oC處理前三天Fv/Fm值顯著下降,且其三天後之Fv/Fm值較30/25o處理者低。由生長表現、葉綠素濃度、葉片相對傷害值及葉綠素螢光值調查項目顯示,Cb5-M612S1比夏蓳‘Clown Rose’有較佳之低溫(15/13 oC)耐受性。
取Cb5-M612S1不同大小花蕾培植體,置於添加不同濃度NAA與BA之1/2MS培養基,結果顯示花萼稜未發育之花蕾培植體所誘導的芽體數較花萼稜完全發育之花蕾培植體多,以花稜尚未發育階段花蕾培植體培養在0.1 mg‧L-1 NAA 組合2 mg‧L-1 BA平均可獲得較多不定芽數目。
取葉片培植體誘導15天發育至心形期體胚,進行分切繼代至含有不同濃度BA及NAA之1/2MS培養基中,培養四個月後調查體胚發育情形,結果顯示各處理都有癒傷組織形成。每個體胚培植體於不添加任何生長調節劑之培養基中平均可再生6.5個體胚苗且均能發根。於0.1mM NAA組合BA培養基誘導下,體胚苗數目隨BA濃度提高而下降。
取培養四週,長0.5-1.0 cm之葉片誘導之除根體胚苗,沾取不同濃度之IBA與NAA發根劑,結果顯示體胚苗於出瓶四週後之發根率以不沾發根劑之對照組最佳,為93%,平均根數3.9條,主根長達3.6 cm。發根率、平均根數、地上部乾重於IBA與NAA發根劑處理間無顯著差異,地下部乾重以NAA處理者較低。
探討不同疊氮化鈉施用方式及處理部位對Cb5-M612之誘變影響。結果顯示於相同濃度疊氮化鈉處理下,以疊氮化鈉培養基處理者,隨培養週數由1週增加至4週,體胚培植體存活率逐漸降低,以疊氮化鈉溶液浸泡處理者,隨浸泡時間由30 min增加至90 min會造成體胚苗數目逐漸下降。比較花蕾培植體與體胚培植體浸泡疊氮化鈉溶液,結果顯示花蕾培植體之存活率隨處理濃度(1、2及4 mM)及浸泡時間(30、60、90 min)增加而顯著降低,顯示花蕾培植體相對於體胚培植體,藥劑傷害程度較大。經疊氮化鈉藥劑處理獲得內輪花序增生(Paracorolla)、雄蕊瓣化、側花瓣分裂之三種重瓣花類型及一株淡花色之變異株。



Abstract
Wishbone flower (Torenia fournieri Linden) is one of the most important summer bedding plants in Taiwan. Torenai fournieri ‘Clown Rose’ is erect and cold-intolerant. Tetraploid Torenia hybrids from crossing T. fournieri ‘Clown Rose’ with T. benthamiana (Cb) are trailing and low-temperature tolerant, as shown by a previous study. Further colchicine treatments resulted in Cb5-M612M1. The objectives of this study were 1) to investigate flower petal traits of selected self-crossed tetraploid Torenia hybrid progeneies (Cb5-M612S1), 2) to establish micropropagation technique using various explant sources and sodium azide treatments, 3) to select new cultivars/ lines.
Flow cytometric analysis revealed that Cb5-M612M1 was approximately tetraploid. The Cb5-M612M1 was then self-crossed to obtain Cb5-M612S1 and Cb5-M612S2 for the measurements of flower traits. Results showed that serrate edge of lateral petal was dominant to whole edge in S1 and S2 populations and population ratio was approximately 15 : 1 (χ2 = 1.65 <χ20.05,2 = 5.99). All plants had papillae petals with yellow dot in S1 and S2 populations. Corolla size of S1 progeneies showed segregation. The selected large-flowered progeny (Cb5-M612S-34) had similar length of stalks, bracts, and corollas as compared to T. fournieri ‘Clown Rose’ .
Cb5-M612S1 and T. fournieri ‘Clown Rose’ were grown under day/night temperatures of 30/25 and 15/13 oC for 21 days. Cb5-M612S1 at 15/13 oC all survived and maintained high SPAD-502 value, while 50% of T. fournieri ‘Clown Rose’ at 15/13 oC died. Leaf relative injury value in Cb5-M612S1 was lower than T. fournieri ‘Clown Rose’ both under 30/25 and 15/13 oC. Fv/Fm value of Cb5-M612S1 did not differ between temperatures, while T. fournieri ‘Clown Rose’ had significantly decreased Fv/Fm value at first three days at 15/13 oC and thereafter remained lower Fv/Fm values than plants at 30/25 oC. In conclusion, Cb5-M612S1 showed better low temperature (15/13 oC) tolerance than T. fournieri ‘Clown Rose’ according to growth performance, leaf chlorophyll concentration, leaf relative injury, and leaf Fv/Fm value.
Different sizes of flower bud explant in Cb5-M612S1 were cultured on 1/2 MS medium containing various NAA and BA concentrations. Results showed that young flower buds with folded calyx developed more adventitious shoots than those mature flower buds. Young flower buds with folded calyx cultured on 1/2 MS supplemented with 0.1 mg‧L-1 NAA+ 2 mg‧L-1 BA had higher shoot regeneration.
Heart-shaped somatic embryos induced from the leaf explant of Cb5-M612M1 were used for somatic emblings multiplication. Each somatic embryo explant produced an avearge of 6.5 somatic emblings and 100% rooting when cultured without plant regulators. Number of somatic emblings decreased when BA concentration increased from 1 mg‧L-1 to 4 mg‧L-1 in medium supplemented with 0.1 mg‧L-1 NAA.
After 4 weeks in vitro culture, somatic emblings with 0.5-1.0 cm length were used for ex vitro rooting experiments. Results showed that somatic emblings without rooting hormone had highest rooting percentage. There were no significant differences in rooting percentage, root number, and shoot dry weight between IAA and NAA treatments. NAA treatments resulted in lower root dry weight.
Sodium azide (SA) treatments and explant source were investigated for the mutation in Cb5-M612M1. Results showed that somatic embryo explant survival percentage decreased when SA medium culture duration increased from 1 to 4 weeks, and somatic emblings number per explant decreased with increasing SA solution soaking duration from 30 to 90 min. Flower bud explants were more sensitive to sodium azide solution soaking treatment than somatic embryo explant. Three double-flowered mutations (paracorolla, petaloid of stamen, and dissection of lateral petal), and one pale flower color mutation were obtained after sodium azide treatments.


目錄
摘要 i
Abstract iii
表目錄 ix
圖目錄 x
前言(Introduction) 1
前人研究(Literature Review) 4
一、倒地蜈蚣屬重要種源 4
二、夏蓳育種史與品種選育方向 5
三、耐寒性狀導入及檢測 6
四、植物色素生合成系統 8
五、夏蓳花色素組成與花色調控 9
六、雜交後代之花朵性狀表現 10
七、重瓣花分類及其調控 11
八、影響重瓣花的外在因素 14
九、多倍體化育種途徑及應用 14
十、微體繁殖再生系統 15
十一、花卉誘變育種 19
材料與方法 (Material and Methods) 22
試驗一、Cb5-M612稔性篩選及其DNA含量分析 22
試驗二、Cb5-M612S1與其來源親本花朵性狀比較及大花單株選拔 23
試驗三、Cb5-M612S1及Cb5-M612S2花朵性狀調查 23
試驗四、日/夜溫對Cb5-M612S1與夏蓳‘Clown Rose’ 葉綠素螢光值、葉綠素計讀值、相對生長速率與葉片相對傷害值之影響 24
試驗五、NAA與BA對Cb5-M612不同大小之花蕾培植體誘導再生不定芽之影響 25
試驗六、NAA與BA對Cb5-M612葉片衍生體胚誘導再生之影響 26
試驗七、IBA與NAA濃度對Cb5-M612體胚苗瓶外發根之影響 26
試驗八、疊氮化鈉培養基濃度與處理時間對Cb5-M612體胚存活率、再生體胚苗數目與誘變率之影響 27
試驗九、疊氮化鈉浸泡濃度與時間對Cb5-M612體胚培植體存活率、再生體胚苗數目與誘變率之影響 28
試驗十、疊氮化鈉浸泡濃度與時間對Cb5-M612花蕾再生不定芽數目、芽體再生率與誘變率之影響 28
結果(Results) 30
試驗一、Cb5-M612稔性篩選及其DNA含量分析 30
試驗二、Cb5-M612S1與其來源親本花朵性狀比較及大花單株選拔 30
試驗三、Cb5-M612S1及Cb5-M612S2花朵性狀調查 30
試驗四、日/夜溫對Cb5-M612S1與夏蓳‘Clown Rose’ 葉綠素螢光值、葉綠素計讀值、相對生長速率與葉片相對傷害值之影響 32
試驗五、NAA與BA對Cb5-M612不同大小之花蕾培植體誘導再生不定芽之影響 32
試驗六、NAA與BA對Cb5-M612葉片衍生體胚誘導再生之影響 33
試驗七、IBA與NAA濃度對Cb5-M612體胚苗瓶外發根之影響 33
試驗八、疊氮化鈉培養基濃度與處理時間對Cb5-M612體胚存活率、再生體胚苗數目與誘變率之影響 34
試驗九、疊氮化鈉浸泡濃度與時間對Cb5-M612體胚培植體存活率、再生體胚苗數目與誘變率之影響 34
試驗十、疊氮化鈉浸泡濃度與時間對Cb5-M612花蕾再生不定芽存活率、芽體再生數目與誘變率之影響 35
討論(Discussion) 69
參考文獻(References) 77
附錄 (Appendix) 93


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