Dissertations / Theses on the topic 'Lilies Genetics'

The ornamental industry has become an important economic force in recent years, in the UK alone this industry is estimated to be around £2.1 billion, while the international trade is around £60-75 billion (Chandler and Tanaka, 2007). The continued success of the floriculture industry depends on the introduction of new species/cultivars with major alterations in key agronomic characteristics, such as resistance to pathogens, novel flower colour and patterns or control of male fertility. Lilium, one of the most important bulbous ornamental crops, is an attractive and popular cut flower. However, the production of vast quantities of pollen that stains easily and is toxic to animals is not always desirable. The control of pollen release without affecting the appearance of the flower is therefore an important breeding goal. Lilium is also susceptible to several fungal pathogens, including Botrytis cinerea, which infects leaves, stem and flowers leading to a reduction of yield. New cultivars have tended to rely upon selective breeding as a mechanism for trait development. However approaches that utilise transgenes to manipulate traits of interest provide alternative opportunities for the ornamental industry provided that transformation and regeneration can be achieved efficiently. A rapid, highly efficient and reproducible Agrobacterium-mediated transformation for Lilium has been developed. Successful transient GUS expression in callus, shoots and basal plate discs was achieved using A. tumefaciens strain AGL1 containing plasmid pBI121 harbouring intron-containing GUS and NPTII genes in cultivars "Beverly's Dream", "Star Gazer", "Night Flyer", "Acapulco", "Sweet Surrender" and Lilium leichtlinii. Based on the same transformation protocol, transgenic plants of cv. "Star Gazer" overexpressing the RCH10 chitinase gene from rice were generated. In vitro sporulation assays of these plants showed different levels of resistance to Botrytis cinerea correlated to the level of relative expression of the transgene. This is the first report of induced pathogen resistance in any Lilium cultivar by transgenic approach. Experiments were also conducted to modify fertility and pollen release in Lilium by translating regulatory gene information from Arabidopsis to Lilium. Transgenic plants of cv. "Star Gazer" either overexpressing or silencing the AtMYB26 gene, were generated. RNAi lines showed a delay in anther dehiscence suggesting that pollen development pathways could be conserved between Arabidopsis and Lilium. In addition, partial sequences of the putative orthologues of AtMS1 and AtMYB26 in Lilium were identified and cloned for future research.

Abstract Biotechnology has become a necessity, not only in research, but also in the culture and breeding of lilies. Various methods in tissue culture and molecular breeding have been applied to the production of commercially important lily species and cultivars. However, scientific research data of such species and varieties that have potential in the northern climate is scarce. In this work, different biotechnological methods were developed and used in the production and culture of a diversity of lily species belonging to different taxonomic groups. The aim was to test and develop further the existing methods in plant biotechnology for the developmental work and the production of novel hardy lily cultivars for northern climates. Most of the plant material was started from seeds, which provided genetic variability and new material for breeding. Different features in seed structure were studied with light microscopy and SEM, and different parameters affecting germination were tested. Several tissue culture protocols were also compared with different species using both solid and liquid media. Molecular biological methods were used in assessing genetic background of traditionally grown lilies. Somatic embryogenesis in callus differentiation of callus cultures was studied, and gene expression behind differentiation processes was analyzed with various molecular biological methods. Particle bombardment system was used in genetic transformation. In addition, protoplast isolation methods from various tissues were tested. The main results indicate that many tissue culture methods can be used in research and in mass production with all tested species. Especially in a large-scale production, temporary immersion system is promising. In addition to the conventional bulb scale material, seeds were found to be a suitable starting material for genetic variability required for production of new cultivars, and in the preservation of natural populations. RAPD techniques proved a suitable method for revealing phylogenetic relations of different lily species and cultivars. Methods in DNA and RNA isolation, cloning and analysis were optimized for lily material. In addition, particle bombardment system was successfully used for genetic transformation of lily callus. In the future, more information is needed to understand better the germination and differentiation processes, focusing especially in the genes, their products and function. In addition, the large and still mostly unknown lily genome is a challenge for research in the future. However, the currently presented results provide good opportunities for further developmental work and research of hardy lily species.

碩士
國立臺灣師範大學
生物學系
84
The population of Lilium longiflorum along eastern coast of Taiwan always occurs in isolated habitats.The population genetic structure of a species which is island distribution like L. longiflorum is a good theme to study.Thirteen population weresampled and genetic variation was investigated using starch gel eletrophoresis.Genetic disversity was rich for the species( Hes=0.191)as well as within population(Hep=0.142).Of the 20 allozyme loci examined,13(65%)were polymorphic,and on average35.38% of the loci were polymorphic within population.The mean number of allelesper locus was 1.61 and the mean effective number of that was 1.17 within population.We found a high proportion of total genetic variation (24%) among population and a significant correlation (p<0.001) between genetic distance and geographic distance.Gene flow and nature selection appear to play major roles in the population structureof the species,while gene folw is more influential than nature selection.

碩士
國立中興大學
農藝學系
94
Summary The main objectives of the present study were: (1) to standardize a protocol for transfer of foreign genes into maize (Zea mays. L.) and Lilium spp. by using a mixture of Agrobacterium tumefaciens strain EHA105 harboring a standard binary vector pCAMBIA1302 and pollen. (2) to investigate the factors like concentration of Agrobacterium and acetosyringon (AS), and duration of co-culture for efficiency of transformation (3) to confirm the transformation events by detection of GFP in transgenic plants by fluorescence microscope; and other molecular techniques like PCR, PCR-Southern and Southern. (4) to analyze transgenic plants for flavonoids by TLC and HPLC. The results obtained during the course of the study are as follows: (1) Protocols of genetic transformation in maize (Zea mays. L.), Lilium longiflorum and Lilium formosanum with Agrobacterium tumefaciens mediated T-DNA delivery system were standardized. In maize, Agrobacterium at 0.45×108 CFU/ml, acetosyringon (AS) at 1 mM, 6 hours co-cultivation of pollen and Agrobacterium were found optimum for transformation. The mixture of Agrobacterium and pollen was used for pollination. After 45-60 days of pollination, seeds obtained were kept at selection pressure of hygromycin at 20 mg/L. Using optimum conditions, it was observed that each pollination treatment in maize resulted into an average of 0.66 plant surviving selection pressure. In Lilium longiflorum, Agrobacterium concentration at 9×108 CFU/ml and AS concentration at 1 mM and 0 hours co-cultivation with pollen, resulted in an average of 2.67 plants surviving selection pressure per pollination treatment. While in case of Lilium formosanum, Agrobacterium concentration at 0.45×108 CFU/ml and AS concentration at 1 mM and 24 hours co-cultivation with pollen, resulted in an average 2.33 plants surviving selection pressure per pollination treatment. (2) Molecular analysis to confirm transformation: In maize, 23 hygromycin -resistant plants were obtained after one month at selection pressure. PCR analysis showed that out of 13 transgenic lines examined, only 9 contained hptⅡ and gfp genes. The GFP gene was identified by PCR-Southern. Out of 3 PCR positive plants, Southern blot showed only one plant contained hptⅡ and gfp gene in maize. In Lilium formosanum, 30 hygromycin -resistant plants were obtained after two months at selection pressure. PCR analysis showed that out of 13 transgenic lines examined, only 10 contained gfp gene. The GFP gene was identified by PCR-Southern. Out of 5 PCR positive plants, Southern blot showed only two plants contained of gfp gene in Lilium formosanum. In another set of experiment, Lilium formosanum was transformed with Agrobacterium strains containing 3 independent flower color genes (dfr, f3h, chi). 34 hygromycin-resistant plants were obtained after two months at selection pressure under optimum conditions. Ten out of 34transgenic lines confirmed by PCR and PCR-Southern were found to contain dfr gene. Southern blot analysis of 5 PCR positive plants showed that 3 plants had dfr gene fragment. While, 5 out of 34 plants were PCR-Southern positive for f3h gene and one was found to contain f3h gene fragment by Southern blot analysis. One out of 34 hygromycin-resistant plants, only one showed PCR-Southern positive for chi gene. (3) Detections of GFP in transgenic plant by fluorescence microscope: GFP expression was observed in seed and leaf of transgenic maize and pollen tube, seed, leaf and petal of Lilium formosanum transgenic plant. (4) The flavonoids contents in the transgenic Lilium formosanum bulb and root were determined by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC), Four samples showed myricetin like compounds as detected by TLC, but HPLC analysis did not show presence of such compounds.

碩士
國立中興大學
植物學系
88
In order to understand the ecophysiological characters of Taiwan orgin lilies to adaptation the environment of their habitat. Lilium formosanum Wall., and L. longiflorum were used as matrials. The bulbs were treated with three different temperature, 10 ℃, 18 ℃ or 25 ℃ from May to October. The results showed that at 10℃ and 18℃, L. formosanum emergenced earlier than at 25. When the date of treatment delayed, L. formosanum emergenced faster at 10℃ (32~66 days), slower at 18℃ (101~138 days), and part almost never emergenced, but part emergence within 2 weeks.When treatment at 25 ℃ in October, the results indicated bulbs dormancy release both in high and low temperature. Without high temperature pretreatment, L. longiflorum showed more emergence rate than that of L. formosanum under 25 ℃. L. formosanum with low-temperature treatment in June and July flowering two month later, but which with the same treatment in November and December flowering six month later. L. formosanum needs low-temperature vernalization treatment and long-day interaction, but L. longiflorum would flower with low-temperature vernalization treatment only. This makes the difference of their flowering period. In the RAPD study, 120 primers were screened and among them 7 primers were selected to analyze all of the samples. AMOVA analysis on RAPD data revealed that, of the total variation of the taxon, 3.92﹪was attributable to population differences between Lilium formosanum Wall., and L. longiflorum, 31.94﹪to population differences within species and 64.15﹪to individual differences within populations. The population variance was all shown to be highly significant (p<0.001).We can distinguish the populations between the east and west of Lilium spp. of Taiwan by cluster. According to the weather record from the weather station, there is much rain in autumn-winter and lots of typhoons in summer in the eastern coast. Thus, the reproduction period lily-emergence in autumn, flowering during March to May and fruit during July to August can maintain the stable nutrition growth and avoid the threat of typhoon during the reproduction period. On the contrast, the west lilies emergence delay because of the drying season in autumn and winter. L. formosanum grows in Taimali population similar emergence and flowering habit to L. longiflorum in the eastern coast. This phenomenon implies that the eco-physiological habits of lilies are more related to the environment where they grow than their genetic relationship.

博士
國立臺灣大學
園藝學研究所
92
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.

Le criocère du lis, Lilioceris lilii (Coleoptera : Chrysomelidae), un ravageur de lis et de fritillaires d’origine eurasienne, a été observé pour la première fois en Amérique du Nord en 1943 sur l’Ile de Montréal au Canada. Après y avoir été confiné pendant environ 25 années, ce coléoptère a par la suite progressé rapidement sur le territoire nord-américain. Actuellement, on l’observe dans huit provinces canadiennes et huit états américains. Cette étude a investigué les routes d’invasion utilisées par le criocère du lis au Canada et aux États- Unis avec l’aide de marqueurs génétiques AFLP. Pour ce faire, 516 individus parmi 34 sites en Amérique du Nord et en Europe ont été échantillonnés et analysés. Le premier objectif était de déterminer, en analysant la structure génétique des populations nord-américains, s’il y avait eu une ou plusieurs introductions en provenance d’Europe. Le deuxième objectif était d’identifier l’origine de la ou des populations introduites en Amérique du Nord. Finalement, le troisième objectif consistait à proposer un scénario d’invasion de L. lilii en Amérique du Nord basé sur les données de première mention et de structure génétique des populations échantillonnées. Les résultats démontrent une signature génétique distincte entre les criocères du lis du Canada et ceux des États-Unis, suggérant ainsi deux sources d’introductions indépendantes en Amérique du Nord, soit une première introduction à Montréal, Québec, dans les années 1940 et une seconde aux États-Unis au début des années 1990 à Cambridge, Massachusetts. De plus, les deux populations nord-américaines semblent provenir de différentes régions du nord de l’Europe, ce qui est conséquent avec le scénario suggérant deux sources d’introductions indépendantes. Chacune des populations aurait par la suite progressé respectivement dans leur pays d’introduction selon une dispersion de type stratifiée. En effet, la progression continue de L. lilii dans certaines régions suggère une dispersion naturelle de l’espèce sur le territoire nord-américain, alors que la progression rapide sur de longues distances semble être causée par le transport anthropique de lis contaminés.
The lily leaf beetle, Lilioceris lilii (Coleoptera : Chrysomelidae), a Eurasian pest of lilies and fritillaries, was first observed in North America in 1943 on the Island of Montréal, Canada. After being confined to Montréal for approximately 25 years, the beetle quickly progressed in North America, and is currently present in eight Canadian provinces and eight American states. During this study, we have investigated the routes of invasion followed by L. lilii in North America, using AFLP markers. We sampled and analysed 516 individuals from 34 sites across North America and Europe. Our first objective was to characterize the genetic structure of North American L. lilii populations to determine if they originated from a single or form multiple introductions from Europe. The second objective was to identify the geographical origin of the invasive population(s). Finally, the third objective was to trace back the geographical routes of invasion of L. lilii in North America, using information from both the dates of first observations and the genetic structure of sampled populations. Our results showed clear genetic difference between individuals from Canada and the USA, suggesting at least two different sources of introductions of L. lilii in North America. A first episode of introduction took place in Montréal, Canada in the 1940’s while a second introduction occurred in Cambridge, Massachusetts, around 1990. Also, both North American populations seem to originate from different populations in northern Europe, which support the hypothesis of two distinct sources of introduction. Each population next progressed in its respective country following a stratified dispersal. Indeed, continuous progression of the beetle in some regions of North America suggests a pattern of natural dispersion, while human-mediated carrying of infected lilies seemed to be responsible for the long-range movement of the beetle.