原生鐵炮型百合形態變異、遺傳歧異及早熟相關葉片形態之研究

Studies on morphological variation, genetic diversity and the precocity related leaf pattern of Lilium formosanum and L. longiflorum native in Taiwan

戴廷恩(Ting-En Dai)

指導教授 ： 許圳塗

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.01646

摘要

將台灣依離岸中央山脈地形（北部山區、中部山區、南部山區等3區域，共計19位點族群）及海岸離島地形（東北角海岸、東部海岸、離島等3區域，共計13位點族群），針對原生台灣百合及鐵炮百合以收集、保存，調查兩種間及族群間形態特性分析，並進行離場溫室栽培保存及體外限制生長保存，以做為原生種保育及於育種上利用之參考。
原生百合植株高度變化大，32位點族群總平均株高由12.6±7.8公分（澳底）至135.0±49.4公分（羅娜）。離岸中央山脈19個族群其總平均株高為71.43±40.43，最高135.0±49.4公分（羅娜），最小21.4±5.7公分（合歡山），株高形態於族群內及族群間均呈高度變異性。海岸離島13個族群其總平均株高為32.84±17.25，最高51.8±23.0公分（金山），最小12.6±7.8公分（澳底），同樣顯示株高呈高度變異。
葉長寬比值則呈規則性變化，32個族群以18-20為界分為兩群，20以上的有19個族群，比值18以下的有13個族群。離岸中央山脈北中南山區19個族群平均葉長寬比值為24.44±6.78，最大34.4±11.4（觀霧），最小20.1±4.7（瑪家）。海岸及離島13個位點族群平均比值為12.28±5.46，最大18.4±4.4（磯碕），最小7.0±1.5（八斗子）。商業栽培種則呈小葉長寬比值（8.6±1.6）。
本研究結果顯示（1）離岸中央山脈原生台灣百合族群之形態發育可塑性大於海岸及離島區之原生鐵炮百合族群，（2）葉長寬比：18-20，可為初步區分台灣百合及鐵炮百合之依據，（3）利用半量營養培養基及低溫限制營養生長，繼代培養時間可以延長到1年半以上，種原材料仍然保有活性。

將台灣依離岸中央山脈地形（北部山區、中部山區、南部山區等3區域，共計19位點族群）及海岸離島地形（東北角海岸、東部海岸、離島等3區域，共計13位點族群），每一個位點族群取樣15株，總計32位點族群480株，利用分子標誌分析探討台灣百合及鐵炮百合兩種間之遺傳歧異及族群之變異特性，以做為原生種遺傳資源保育及於育種上利用之參考。
利用8對18至26-Base微衛星體引子（Y01、Y13、Y18、Y22、RM8、RM17、RM22及CT58）及4個10-Base 逢機引子（A10、A18、H4、J19）進行RAPD反應。參計32族群480株，共得到86個增殖DNA片段（markers），每個引子（對）產生5-14個條帶（平均產生7.2個條帶），表現84.38％多型性。就以Y01引子對為例，可產生14個不同條帶片段，各族群之主要條帶佔50.4％左右，其餘為次要條帶及稀有條帶，顯示族群內具高度變異。86個條帶中共出現10個特異性條帶，台灣百合及鐵炮百合分別有7個及3個族群特異性條帶。
採用Nei（1972）所提出之相似度公式來計算不同族群間之遺傳距離指標 （Index of Genetic Distance），族群間遺傳距離以新望洋（SWY）及霧社（WUS）族群最為接近（IGD=0.0184），蘭嶼（LA）及屯原（TU）族群遺傳距離最遠（IGD=0.1746）。UPGMA分析結果除觀霧（GW）族群與合歡山（HH）族群相近而被歸類到中部山區之外，其餘族群均完全對應到取樣區域而互相歸類在一起，同時可區分為離岸中央山脈區的百合群及（台灣百合）及海岸離島區的百合群集（鐵炮百合）兩大類。AMOVA分析原生鐵炮型百合族群間的變方成份為3.645（32％），族群內個體間的變方成份為7.882（68％）。中高海拔台灣百合及海岸低海拔鐵炮百合族群區域間變方成份為2.443（19％），區域內族群間的變方成份為2.482（19％），族群內個體間的變方成份為7.893（68％）。
本研究結果顯示選擇之引子及方法足以對32個台灣原生鐵炮型百合族群作完整的分群，可將台灣百合及鐵炮百合配合地理區域加以區分。由分子標誌數據之遺傳關係圖及主成分分析結果對照形態標誌，更可確定葉長寬比：18-20，可為初步區分台灣百合及鐵炮百合之依據。
原生鐵炮型百合參試台灣百合及鐵炮百合分別有6個及7個族群樣品，經由體外微體繁殖保存小植株，剝其小鱗片為培殖體，扦插於再生培養基中，比較其不定芽發生增殖及鱗片葉發生效率。
參試品系小鱗片扦插再生不定芽數，品系間變化為1.33至2.67個。鐵炮型百合不定芽朝向小鱗莖生長，除第一枚葉原體呈無葉身的鱗片型（scale type）生長外，後續的葉原體則朝向有葉身之鱗片葉型（scale leaf type or foliage scale）生長為主。對照東方型百合栽培種’Casa Blanca’小鱗莖形成，則以鱗片型生長為主，可歸之為無綠葉小鱗莖（Non-green leaf bulblet）。
於再生培養基12週所形成鱗片葉數，參試台灣百合及鐵炮百合品系間變化為4.28至7.06片。FXCA及LXCA亦呈鱗片葉型結球，其鱗片葉數分別為4.67及3.89片，較母本為低，但遠高於父本。再生小鱗莖分株繼代培養12週，原生鐵炮型百合品系間鱗片葉數目變化為12.22至21.11片，台灣百合及鐵炮百合葉片展開速率平均分別約為每週1.00及0.83片，顯示台灣百合生長勢較鐵炮百合稍強。

微體繁殖體外小鱗莖生長檢定出台灣百合（F）、鐵炮百合（L）為鱗片葉型（scale leaf type）生長，東方型百合栽培種’Casa Blanca’（CA）為鱗片型（scale type）生長，而FXCA及LXCA則呈中間型生長勢之鱗片葉型生長
外加ABA及Fluridone培養4週及8週，鱗片葉生長調查結果顯示（1）各基因型的鱗片葉生長均明顯受到ABA的抑制及Fluridone的促進。（2）ABA會抵銷Fluridone的效應。（3）Fluridone處理促使鱗片葉數增加，但出現白化現象。所有外加ABA及Fluridone的處理，L/O雜交種鱗片葉生長均呈現中間型表現，不論是ABA的抑制生長或Fluridone的促進生長。只有在ABA與Fluridone同時加入培養4週時，L/O雜交種鱗片葉生長表現優於2親本。
ABA濃度變化結果顯示（1）CA有最高內生ABA濃度，F及L最低，L/O雜交種則介於兩親本間。（2）外加ABA促使鱗片ABA濃度增加。（3）Fluridone處理使鱗片ABA內含量濃度減少。（4）同時外加ABA會抵銷Fluridone的作用，促使鱗片ABA內含量濃度增加。
糖對鱗片葉形態發育的調查數據顯示（1）高濃度糖抑制鱗片葉的發生，但抑制效果不如外加ABA，依然有一定程度的鱗片葉發生。（2）高濃度糖同時外加Fluridone的栽培環境，對鱗片葉發生有促進的趨勢，同時使鱗片葉出現紅色（圖5、6、7）。
高濃度糖相關處理間的鱗片ABA濃度變化結果顯示（1）F、L及CA並未因高濃度糖的培養環境而使鱗片內生ABA濃度出現明顯變化。（2）L/O雜交種則不論有無Fluridone存在，高糖濃度均使得鱗片內生ABA濃度出現明顯增加的趨勢。

利用台灣百合（F）、鐵炮百合（L）、東方型百合（CA）及其L/O種間雜交種台灣百合X東方型百合（F X CA）及鐵炮百合X東方型百合（L X CA），研究百合小鱗莖（Bulblet）再生及葉片發生與內生ABA之關係。內層鱗片平均再生約1.67至2.56個小鱗莖，外層鱗片平均再生約2.89至4.22個小鱗莖，顯示外層鱗片的小鱗莖再生力較內層鱗片高。內層鱗片ABA含量平均約2-10 nmol / FW(g)，外層鱗片ABA含量平均約0.5-3 nmol / FW(g)，不同基因型百合內外層鱗片的ABA含量變化呈現相反趨勢。
F培養4週及8週的葉片發生速度並無差異，每週平均約0.7片，培養時間至12週時，每週平均生長約1片葉。L及L/O種間雜交種（F X CA、L X CA），葉片發生速度隨培養時間增加而有降低之趨勢，培養12週後，平均總葉片生長速度分別為每週0.87, 0.35及0.27片。CA培養時間4至12週之葉片發生速度並無明顯差異。葉片發生速度，再生總葉片數及再生總鱗片葉數，均以鐵炮型百合（F、L）表現最快，東方型百合（CA）最慢，L/O種間雜交種（F X CA、L X CA）則呈現中間型表現。鐵炮型百合（F、L）的ABA含量，大致上均小於1 nmol / FW(g)， CA的ABA含量最高，約2.7至3.3 nmol / FW(g)，F X CA及L X CA的ABA含量分別約在1.0至2.0 nmol / FW(g)及1.5至3.0 nmol / FW(g)之間，均呈現中間型表現，介於兩親本間。
每週平均總葉片生長速率，受到外加ABA之影響而明顯降低，培養4週後，平均降低19％（L X CA）至52％（F X CA）。移除ABA，再以基本培養基再培養4週，總葉片生長速率便會恢復，甚至於高於ABA處理之前（F、CA、L X CA）。培養基中加入ABA（1 mg/L），培養4週後，使百合鱗片ABA含量急速上升，大幅增加，F增加約20倍。移除ABA， 培養4週後，百合鱗片ABA含量明顯下降，顯示ABA反應為暫時性反應。

關鍵字

ABA ； 早熟 ； 葉片形態 ； 遺傳歧異 ； 形態變異 ； 台灣百合 ； 鐵炮百合

並列摘要

Different germplasm were collected from geographic districts in “Central Mountain District” (northern area, central area and southern area, 3 areas and 19 population sites) and in “Seashore-Outlying Islets District” (northeastern area, eastern area and outlying islets, 3 areas and 13 population sites). Studying morphological characteristics of different accessions in L. formosanum and L. longiflorum native in Taiwan can provide us the needed information in conservation and breeding program.
A great diversity existed in the characteristic of plant height. The minimum average plant height of 32 populations is 12.6±7.8 cm (Au-Di) and the max is 135.0±49.4 cm (Luo-Na). The average plant height of 19 populations in “Central Mountain District” is 71.43±40.43 cm with the max 135.0±49.4 cm (Luo-Na) and the min 21.4±5.7cm (He-Huan-Shan). The plant height characteristic showed a great diversity in inter- and intra- populations. The average plant height of 13 populations in “Seashore-Outlying Islets District” is 32.84±17.25 cm with the max 51.8±23.0 cm (Jien-Shan) and the min 12.6±7.8cm (Au-Di), showed a great diversity too.
The ratio of leaf length to width showed a regular rule of 18-20 as a margin to separate 32 populations into two groups: 19 populations with a ratio 20 above and 13 populations with a ratio below 18. The total average leaf length/width ratio of 19 populations in “Central Mountain District” is 22.44±6.78, the max. is 34.4±11.4 (Guan-Wu), the min. is 20.1±4.7 (Ma-Ja). The total average leaf length/width ratio of 13 populations in “Seashore-Outlying Islets District” is 12.28±5.46 the max is 18.4±4.4 (Jiyi-Chi), the min is 7.0±1.5 (Ba-Dou-Tz).
The results showed (1) the populations native in “Central Mountain District” (L. formosanum) had more morphological developmental plasticity than the populations native in “Seashore-Outlying Islets District” (L. longiflorum), (2) the ratio of leaf length and width 18-20 can be the first rule to classify L. formosanum and L. longiflorum, (3) restricted vegetative growth by half culture medium and low temperature, subculture process can prolong to 1 even to 1 and half year and the material still with vigor.

Based on two main geographic districts of “Central Mountain District” (northern area, central area and southern area, 3 areas and 19 population sites) and of “Seashore-Outlying Islets District” (northeastern area, eastern area and outlying islets, 3 areas and 13 population sites) , 15 plants were sampled for each population from 32 populations. The genetic diversity and variance of L. formosanum and L. longiflorum native in Taiwan through molecular markers were analyzed. This information then can be used as a foundation of germplasm conservation and breeding works.
Using 8 pairs microsatellite primers and 4 arbitrary oligonucleotide 10-Base to carry on PCR reactions. 86 fragments were amplified in 480 plants of 32 populations with an average of 7.2 per primer (pairs). Among them, 84.38％ polymorphism were observed. For example, Y01 primer can produce 14 fragments with 50.4％ main fragments and the rest are sub- or rare- fragments, so high variance in intra-populations. Ten specified fragments obtained in 86 amplified fragments, 7 and 3 fragments for L. formosanum and L. longiflorum, respectively.
The IGD (Index of Genetic Distance) between germplasm was calculated by Nei’s (1972). The most closet is Shien-Wuan-Yiang and Wu-She (IGD=0.0184) and the max. is Lan-Yu and Tuen-Yuan (IGD=0.1746). UPGMA results showed, with the exception of Guan-Wu, populations can be distributed together associated with their geographic areas and be divided into “Central Mountain District” populations (L. formosanum) and “Seashore-Outlying Islets District” populations (L. longiflorum). The results of AMOVA of native populations indicated that 32% of the total variation was attributable to the differences among provenances while 68% was due to the variation among individuals within provenances. The results of AMOVA between middle-high attitude and sea shore, low attitude populations indicated that 19% of the total variation was attributable to the differences among regions, 19% was due to the variation among populations within regions and 68％ was due to the variation among individuals within populations/regions.
The results of this study showed, the primers and methods can have a fully categorized of 32 lily populations native in Taiwan and in accord with their geographic regions. The results of UPGMA genetic relation tree based on molecular markers compared with the data of morphological traits, we can make sure that the ratio ranged from 18 to 20 of leaf length and width can be the rule of classification of L. formosanum and L. longiflorum.

L. formosanum and L. longiflorum had six and seven population samples for testing, respectively. Plantlets were conserved and propagated in vitro and be the materials of scaling to compare the efficiency of adventitious buds multiplication and scale leaves development.
Twelve weeks after cutting, the average adventitious buds ranged from 1.33 to 2.67. The adventitious buds of the longiflorum type lilies were tended to be bulblets. The first leaf primordia developed to be scale type, the other primordium will develop to be scale leaf type or foliage scale. The oriental type cultivar ‘ Casa Blanca’ as a contrast, the formation of bulblets was scale type mainly and can group under non-green leaf bulblet.
Twelve weeks after cutting, the average scale leaves of L. formosanum and L. longiflorum ranged from 4.28 to 7.06. FXCA and LXCA showed scale leaf type development and the average scale leaves was 4.67 and 3.89, respectively, lower than female and higher than male parent.
Twelve weeks after subculturing, the scale leaves of longiflorum type lilies ranged from 12.22 to 21.11. The leaf unfolding of L. formosanum and L. longiflorum was 1 and 0.83 per week, showed the former had a stronger vigor.

Exogenous ABA and Fluridone were used to investigate the development of scale leaves. Four and eight weeks after culturing, the results showed that (1) the scale leaves growth of all genotype were remarkably promoted by ABA and inhibited by Fluridone, (2) ABA can diminish the effects of Fluridone, and (3) Fluridone treatments increased the number of scale leaves but bleaching.
All the treatments of exogenous ABA and Fluridone, L/O distant hybrids showed intermediate performance, the inhibition effects of ABA and promotion effects of Fluridone all have the same trends. Only exogenous ABA and Fluridone were added together, after 4 weeks, the scale leaves growth of L/O hybrids had superior performance than their parents. The results of ABA concentration showed that (1) CA have the highest endogenous ABA concentration, the lowest in the F and L and L/O hybrids with intermediate ABA content between the two parents, (2) exogenous ABA stimulated the increasing of scale endogenous ABA concentration, (3) Fluridone diminished the scale endogenous ABA concentration, and (4) exogenous ABA can diminished the effects of Fluridone and increasing the scale endogenous ABA concentration.
To investigate the effects of sucrose on the scale leaves morphogenesis showed the results (1) High concentration sucrose inhibited the development of scale leaves, but the degree of inhibition was less than exogenous ABA, still having little scale leaves to emerge, (2) in the environment of high concentration sucrose added Fluridone had the trend to promote the scale leaves development and with red color.
From the effects of related treatments of high concentration sucrose on the scale ABA, the data showed (1) cultured under the high concentration sucrose condition, F, L and CA did not have remarkable change on the scale endogenous ABA, (2) high concentration sucrose will have the trends to increasing the scale endogenous ABA of L/O hybrids, no matter Fluridone existed whether or not.

Lilium formosanum (F), L. longiflorum (L), Oriental type of lily (CA) and two hybrids (F x CA and L x CA) were used to investigate the relationship between scale propagation, scale leaf development and endogenous ABA. Inner-scales generated about 1.67-2.56 and outer-scales generated about 2.89-4.22 bulblets, respectively. Outer-scales got higher ability to propagate bulblets than the inner-scales. The concentration of ABA in Inner-scale is about 2-10 nmol/FW(g) and outer-scale is about 0.5-3 nmol/FW(g) respectively.
Four and eight weeks after culture, the average formation rate of leaves of F is about 0.7 per week, and with the rate was up to 1 per week after 12 weeks. The average formation rate of leaves reduced after 12 weeks culture in L and two interspecific hybrids (F x CA and L x CA). It was 0.87 per week for L, 0.35 for F x CA, and 0.27 for LxCA. There is no difference in the rate of leaf formation from 4 to 12 week culture in CA. The F and L had the highest rate in leaves formation, total leaf number and scale leaves. Oriental type of lily (CA) had the lowest rate while two interspecific hybrids (F X CA and L X CA) had the medium rate. The concentration of ABA in was less than 1 nmol/ FW(g) in the Longiflorum Type of lily (F and L), about 2.7-3.3 nmols/FW(g) in CA, 1.0-2.0 nmols/FW(g) in F x CA and 1.5-3.0 nmols/FW(g) in L X CA. It is obvious that two hybrids expressed medium concentration of ABA as compared with their parents.
The rate of leaf formation was reduced when the exogenous ABA was applied to the plantlets. After 4 weeks culture, the rate reduced from 19% (L X CA) to 52% (F X CA). After ABA removed, the rate of leaf formation was back to normal or higher in the some populations (F, CA and L x CA). Adding ABA (1 mg/L) in the culture medium for 4 weeks will significantly raise the concentration of ABA in the scales about 20 times in F. Four weeks after ABA removal, the concentration of ABA was significantly reduced. Therefore, the ABA effects can be reversed in this study.

並列關鍵字

L.formosanum ； leaf-morphology ； ABA ； precocity ； genetic-diversity ； morphological-variation ； L.longiflorum

參考文獻

柯見螢、楊堯文、陳福旗. 2003. 利用逢機擴大多型性DNA (Random Amplified Polymorphic DNA, RAPD) 標記探討白鶴芋品種之遺傳變異性. 中國園藝49(1): 45-53.

翁仁憲. 1999. 台灣原生百合之種子發芽對溫度之反應. 中華民國作物種原簡訊4(1): 1-5.

翁儷倩、楊雯如、張龍生、林宗賢. 桶柑遺傳歧異性之分析. 中國園藝49(1): 19-31.

許圳塗. 1977. 臺灣原生鐵炮百合 (Lilium longiflorum Thunb.) 及臺灣百合 (L. formosanum Wall.) 不同族群結實及種子發芽特性. 中國園藝23(1): 15-22.

鄭免、許圳塗. 1984. 苗期溫度、低溫及光週處理對台灣百合抽莖及開花的影響. 中國園藝30 : 50-58.