臺灣博碩士論文加值系統

本實驗之目的為利用生育性狀、RAPD標記、體胚發生和核型分析技術，建立白鶴芋 (Spathiphyllum) 品種遺傳變異評估的方法。將十二個白鶴芋品種或種分析其形態性狀，結果顯示有顯著的差異，有些品種可藉由株高、葉數和側芽數等性狀來區別，但大多數品種無法完全鑑定區別。供試品種或種經根尖染色體數檢查的結果顯示，S. 'Max'與S. 'Sensation'品種的染色體數均為2n = 45，屬於三倍體，其他品種如S. 'Pallas'、S. 'Luna'、S. 'Ceres'、S. 'Stephanie'、S. 'Illusion'、S. 'Vanessa'、S. 'Domino'、S. 'Jetty'、S. 'Petite'、S. cannifolium均為2n = 30，屬於二倍體。供試白鶴芋屬品種或種大部份為中部著絲點染色體 (m) 為主，至少具有兩對近中部著絲點染色體 (sm)，三倍體品種均有4對近中部著絲點染色體 (sm)。核型分析結果顯示，S. 'Max'和S. 'Sensation'的核型公式完全相同，皆是2n = 45 = 33 m + 12sm。其它品種如S. 'Luna'、S. 'Petite'、S. 'Jetty'和S. 'Stephanie'具有相似核型都為2n = 30 = 24 m + 6 sm，而S. 'Petite'第三條染色體具有次縊痕結構，S. 'Ceres'、S. 'Domino'、 S. 'Vanessa'、S. 'Illusion'、S. 'Pallas'和S. cannifolium具有相似核型都為2n = 30 = 26 m + 4 sm，但S. cannifolium第三條染色體具有次縊痕結構。分析最長和最短染色體長度比值介於1.33-1.76之間。品種間核型對稱係數分佈在56.48-59.70 %之間，顯示其核型對稱程度高。依據Stebbin核型分類，S. cannifolium核型類型為1A，其它品種核型類型均為2A。
以95組隨機引子進行RAPD分析，其中有14組引子在PCR反應後，在電泳膠片上可產生多型性清楚DNA條帶，此14組引子產生的243個DNA條帶 (RAPD marker)，有92條為單一個品種所特有，此包含41個為S. cannifolium所特有，4條帶為所有供試品種所共有，147個DNA條帶均為多型性的條帶，可以用來分辨不同的品種。利用Jaccard (1908) 和Nei & Li (1979) 所提出的相似度 (similarity coefficient)公式來計算出不同品種之間遺傳相似性及UPGMA方法所建立出來的親緣關係圖，分析結果顯示，S. cannifolium和其它品種差異最遠，其它品種如S. 'Max'、S. 'Pallas'、S. 'Luna'、S. 'Ceres'、S. 'Stephanie'、S. 'Illusion'、S. 'Sensation'、S. 'Vanessa'、S. 'Domino'、S. 'Jetty'和S. 'Petite'為一群，此群又可明顯分為兩群，其中S. 'Max'與S. 'Sensation'為接近一群，其餘品種為相似一群。因此這些引子可以做為品種鑑定的標記以及品種間遺傳變異性之評估。
以白化苗節間培養於不同濃度2,4-D和NOA組合之培養基中，探討對S. 'Sensation'體胚發生之影響，結果顯示除了N2-i和N2-m組合無法產生體胚外，其餘組合均可產生體胚，其發生頻率在5.0-26.7%之間。癒合組織發生頻率在47.5-82.5 %之間。以N2-n (0.1 mg/l 2,4-D和0.02 mg/l NOA) 和N2-b (0.01mg/l 2,4-D和0.02 mg/l NOA) 兩種組合之培養基來比較S. 'Sensation'、S. 'Ceres'、S. cannifolium、S. 'Pallas'、S. 'Vincent'和S. 'Enchantment'等六個品種體胚發生之能力，結果顯示此兩種組合之培養基對六個品種都能誘導體胚，其中N2-n 組合之培養基，除了S. cannifolium外其餘五個品種皆可產生，以S. 'Ceres'發生的頻率最高為25.0 %，而S. 'Vincent'發生的頻率最低為7.5 %。而N2-b組合之培養基，除了S. 'Pallas'外其餘五個品種皆可產生，其中以S. 'Enchantment'產生的頻率最高為30.0 %，而S. 'Ceres'發生的頻率最低僅5.0 %。癒合組織發生頻率在47.5-82.5 %之間。將體胚移至不含生長調節劑的培養基中，可以發育成完整的植株，利用石蠟切片觀察印證為體胚構造。

The purposes of this study are to evaluate genetic variation in Spathiphyllum germplasms based on morphological traits, RAPD markers, somatic embryogenesis and karyotype analysis. We examined morphological traits, such as leaf length, plant height and leaf numbers of 12 Spathiphyllum cultivars. The results showed that morphological traits were significantly different among cultivars. Both S. 'Max' and S. 'Sensation' were shown to be triploid with 2n = 45. Other cultivars, including S. 'Pallas', S. 'Luna', S. 'Ceres', S. 'Stephanie', S. 'Illusion', S. 'Vanessa', S. 'Domino', S. 'Jetty', S. 'Petite' and S. cannifolium were shown to be diploid with 2n = 30. Most genomes are with median type (m) and at least two pairs of submedian (sm) chromosomes, while two triploid cultivars having four pairs of submedian chromosomes. The karyotype of both S. 'Max' and S. 'Sensation' are 2n = 45 = 33m + 12sm . The karyotypes of other cultivars, including S. 'Luna', S. 'Petite', S. 'Jetty' and S. 'Stephanie' are 2n = 30 = 24 m + 6 sm, but the S. 'Petite' has secondary constriction in the 3rd chromosome. The karyotypes for S. 'Ceres', S. 'Domino', S. 'Vanessa', S. 'Illusion', S. 'Pallas' and S. cannifolium are 2n = 30 = 26 m + 4sm； however, S. cannifolium has a secondary constriction in 3rd chromosome. The ratio for longest and shortest chromosome is between 1.33 and 1.76. The centromeric terminalization value of chromosomes is 56.48-59.70 %, which shows high symmetry of karyotype in the genus. According to Stebbins (1971), karyotype for S. cannifolium was classified to 1A, while karyotypes of other cultivars were classified to 2A.
Fourteen out of 95 random primers were selected in PCR reaction after preliminary screening. A total of 243 RAPD markers were amplified among these cultivars. Among them, 92 are cultivar specific, including 41 bands in S. cannifolium. Four bands are monomorphic for 12 cultivars, and 147 are polymorphic. Using UPGMA for dendrogram construction, S. cannifolium was shown to be most distant form other cultivars. All other cultivars can be subdivided into two groups with S. 'Max' and S. 'Sensation' in a group and others in the other group.
Etiolated internode explants were cultured on 2,4-D and NOA containing MS media to induce somatic embryogenesis of S. 'Sensation'. The results showed that somatic embryos can be induced in these media except N2i and N2-m media. The frequency of somatic embryo formation rate was between 5.0%-26.7%. Callus formation rate was also between 47.5-82.5%. N2-n and N2-b media were used to compare somatic embryogenesis of six cultivars. The results showed that N2-n (0.1 mg/l 2,4-D and 0.02 mg/l) and N2-b (0.01 mg/l 2,4-D and 0.02 mg/l) media were able to induce somatic embryo formation of six cultivars. The somatic embryo formation rate was 25.0 % for S. 'Ceres', highest among six cultivars. Callus formation rate was between 10-50 %. On N2-b medium, embryo formation rate was 30.0 % for S. 'Enchantment', highest among six cultivars. Callus formation rate was between 12.5-62.5 %. Plantlets can be regenerated after transferring somatic embryos onto MS basal medium. Paraffin sections confirmed somatic embryo structure.

中文摘要------------------------------------------------------------------------------------------------I
英文摘要----------------------------------------------------------------------------------------------III
誌謝-----------------------------------------------------------------------------------------------------V
目錄----------------------------------------------------------------------------------------------------VI
圖次索引--------------------------------------------------------------------------------------------VIII
表次索引----------------------------------------------------------------------------------------------X
前言------------------------------------------------------------------------------------------------------1
前人研究-----------------------------------------------------------------------------------------------2
一、 白鶴芋之分類地位及特徵---------------------------------------------------------------2
二、 白鶴芋染色體數----------------------------------------------------------------------------2
三、 核型分析---------------------------------------------------------------------------------------3
四、 品種鑑定之技術----------------------------------------------------------------------------4
五、 體胚發生--------------------------------------------------------------------------------------14
材料與方法------------------------------------------------------------------------------- -----------18
一、 植物材料--------------------------------------------------------------------------------------18
二、 生育性狀調查-------------------------------------------------------------------------------18
三、 根尖染色體數調查------------------------------------------------------------------------18
四、 核型分析的操作---------------------------------------------------------------------------19
五、 RAPD-PCR分析-----------------------------------------------------------------------------20
六、 核酸條帶資料分析------------------------------------------------------------------------22
七、 體胚發生--------------------------------------------------------------------------------------22
結 果---------------------------------------------------------------------------------------------------26
一、 生育性狀調查-------------------------------------------------------------------------------26
二、 根尖染色體數-------------------------------------------------------------------------------29
三、 染色體核型分析----------------------------------------------------------------------------29
四、 白鶴芋的品種鑑定及親源關係-------------------------------------------------------32
1、 隨機引子篩選---------------------------------------------------------------------------------32
2、 RAPD標記分析--------------------------------------------------------------------------------33
3、 群叢分析----------------------------------------------------------------------------------------37
五、 體胚發生---------------------------------------------------------------------------------------39
1、 不同濃度2,4-D和NOA組合對S. 'Sensation'再生之影響-------------------------39
2、 不同品種間體胚發生之差異性---------------------------------------------------------40
3、 體胚發生之組織切片觀察----------------------------------------------------------------40
討 論----------------------------------------------------------------------------------------------------84
參考文獻-----------------------------------------------------------------------------------------------92

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