Orchid Biotechnology Iii

Wen-Huei Chen, Hong-Hwa Chen

• Publisher: WSPC
• Year: 2017
• eBook ISBN: 9789813109230
• Topic: Biological Sciences
• Subtopic: Ecology
• Index: Biological Sciences

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This book provides a first hand and complete information on orchid biotechnology for orchid lovers, graduate students, researchers and industry growers. It contains comprehensive genomics and transcriptomics data, and a thorough discussion of the molecular mechanism of orchid floral morphogenesis. The contributors to the book are all orchid enthusiasts with more than 20 years' experience in the field.

With more than 25,000 species, orchids are the most species-rich of all angiosperm families. They show wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth. Orchids are fantastic for their spectacular flowers with highly evolved petal, labellum, and fused androecium and gynoecium, gynostemium, to attract pollinators for effective pollination. In addition, orchids have attracted the interest of many evolutionary biologists due to their highly specialized evolution and adaptation strategies.

Orchid Biotechnology III covers the most update knowledge of orchid biotechnology research on Phalaenopsis, Oncidium, Cymbidium, Anoectohilus, Paphiopedilum, and Erycina pusilla. It will provide graduate students, researchers, orchid lovers and breeders with an opportunity to understand the mechanism why the orchids are so mysterious and spectacular. Hopefully, this information will be helpful for breeders to enhance orchid breeding and create even more elegant and grace flowers.

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Contents: Genome Size Variation in Species of the Genus Phalaenopsis Blume (Orchidaceae) and Its Application in Variety Improvement (Wen-Huei Chen and Ching-Yan Tang);Distinct Distribution Patterns of 45S rDNA and 5S rDNA-NTS–Related Repeats Display Diverse Karyotypes in Paphiopedilum (Yung-I Lee, Fang-Chi Chang, and Mei-Chu Chung);Chromosome Number Variation in the Emergent Orchid Model Species Erycina pusilla (Hsuan-Yu Yeh, Choun-Sea Lin, and Song-Bin Chang);Comparative Chloroplast DNA Analysis of Phalaenopsis Orchids and Evaluation of cpDNA Markers for Distinguishing Moth Orchids (Ching-Chun Chang, Wen-Luan Wu, Yu-Chang Liu, Cheng-Fong Jheng, Tien-Chih Chen, Bo-Yen Lin, Jhong-Yi Lin, Ting-Chieh Chen, and Yuech-Feng Lee);Development of SSR Markers in Phalaenopsis Orchids, Their Characterization, Cross-Transferability and Application for Identification (Yu-Lin Chung, Yi-Tzu Kuo, and Wen-Luan Wu);Warm-Night Temperature Delays Spike Emergence and Alters Carbon Pool Metabolism in the Stem and Leaves of Phalaenopsis aphrodite (Yo-Ching Liu, Kai-Meng Tseng, Ching-Chia Chen, Yun-Tai Tsai, Cheng-Huan Liu, Wen-Huei Chen, and Heng-Long Wang);Light-Emitting Diodes Affect the Growth and Gene Expression of Phalaenopsis Plantlets In Vitro (Huey-Wen Chuang);Handling, Packaging, and Transportation of Phalaenopsis Plants for Marine Shipment (Chao-Chia Huang, Ching-Chieh Huang, and Pen-Chih Lai);The Orchid-Infecting Viruses Found in the 21st Century (Chia-Hwa Lee, You-Xiu Zheng and Fuh-Jyh Jan); Phalaenopsis Viruses and Their Detection Techniques (Chin-An Chang);Virus Resistance in Orchids (Kah Wee Koh and Ming-Tsair Chan);Perianth Regulation in Orchids (Hsing-Fun Hsu and Chang-Hsien Yang);Flower Development of Phalaenopsis Orchids Involves Functionally Divergent B-Class and E-Class MADS-Box Genes (Zhao-Jun Pan, Wen-Chieh Tsai, and Hong-Hwa Chen);The Function of C/D-Class MADS Box Genes in Orchid Gynostemium and Ovule Development (You-Yi Chen and Wen-Chieh Tsai);The Dual Function of Harpin in Disease Resistance and Growth in Phalaenopsis Orchids (Huey-Wen Chuang);Development of a High-Throughput Virus-Induced Gene-Silencing Vector in Phenotypic Screening of Orchids (Ho-Hsiung Chang, Li Chang, Hsiang-Chia Lu, and Hsin-Hung Yeh);Application of VIGS to Floral Gene Function Studies of the Orchid, a Nonmodel Plant (Ming-Hsien Hsieh and Hong-Hwa Chen);Functional Study of Phytoene Synthase by RNAi-Based Downregulation in the Oncidesa Orchid (Chao-Wei Yeh, Jianxin Liu, Chung-Yi Chiou, Chin-Hui Shen, Peng-Jen Chen, Jung-Chung Liou, Chin-Der Jian, Xiao-Lan Shen, Fu-Quan Shen, and Kai-Wun Yeh);Flower Color and Pigmentation Patterns in Phalaenopsis Orchids (Chia-Chi Hsu and Hong-Hwa Chen);Prolonged Exposure to Elevated Temperature Induces Floral Transition via Upregulation of Cytosolic Ascorbate Peroxidase 1 and Subsequent Reduction of the Ascorbate Redox Ratio in the Oncidium Hybrid Orchid (Dan-Chu Chin and Kai-Wun Yeh);Methylation Effect on Chalcone Synthase Gene Expression Determines Anthocyanin Pigmentation in Floral Tissues of Two Oncidium Orchid Cultivars (Dan-Chu Chin and Kai-Wun Yeh); --> -->
Readership: Graduate students and researchers in agricultural sciences (crop sciences, plant biology, biotechnology, genetics & genomics, microbiology & virology.
-->Orchid, Biotechnology, Flower, Genomics, Virus-Induced Gene Silencing, Morphogenesis, Resistance, Transformation, Virus, Database, Chloroplast, SSR Marker, MADS-Box Genes, Ambient Temperature, LED Light, Chromosome, Pigmentation Patterning, Promoters, Redox

• This book provides the first hand and complete information of orchid biotechnology for orchid lovers, graduate students, researchers and industry growers
• It contains comprehensive genomics and transcriptomics data, and thorough discussion of molecular mechanism of orchid floral morphogenesis
• The contributors for chapters are all endeavor in orchid research for more than 20 years

CHAPTER 1

Genome Size Variation in Species of the Genus Phalaenopsis Blume (Orchidaceae) and Its Application in Variety Improvement*

Wen-Huei Chen^† and Ching-Yan Tang

The genome size of Phalaenopsis species of four subgenera with eight sections was estimated by flow cytometry using propidium iodide (PI) and 4,6-diamidino-2-phenylindol (DAPI). Young ovary tissues of the flowers were used for this study. 2C values of 50 species obtained by using the PI fluorochrome were highly variable. These values varied to different degrees within each subgenus/section. The Nuclear DNA contents of species within the sections of Phalaenopsis and Fuscatae were homogenous without significant difference, while those in the sections of Zibrinae and Amboinenses were highly variable. The subgenera Parishianae and Aphyllae and two sections of the subgenus Phalaenopsis (Esmeralda and Deliciosae) showed higher mean 2C values than those of other sections. Intraspecific variations in 2C values were observed in some species. Comparison of the fluorescence ratios of DAPI and PI of the same species showed highly positive correlation between these two sets of data. Furthermore, there was a tendency for the increase in DAPI fluorescence ratios to be higher when the nuclear DNA content of a species increased. Nuclear DNA measurements obtained from PI and DAPI gave further insight into the genomic structure of the Phalaenopsis species. Considering the taxonomic classification and studies on the molecular phylogeny and biogeographic distribution, our data indicate the possibility of different patterns of phylogenetic relationship among different taxonomic groups of Phalaenopsis. The information provided by this study could also be useful for orchid breeders to enhance the efficiency of their improvement programs.

1.1.Introduction

The genus Phalaenopsis Blume (Orchidaceae), commonly called the moth orchid, comprises five subgenera with approximately 63 species (Christenson, 2001; Tsai, 2011). All diploid species of this genus have the same chromosome number (2n = 2x = 38) (Christenson, 2001). Cytological studies revealed that chromosome sizes are different among species of Phalaenopsis, ranging from 1.5 to 3.5 μm (Shindo and Kamemoto, 1963; Kao et al., 2001). For the development of new varieties of this group of orchids, wild species are often used as one of the parental plants through the process of interspecific hybridization and progeny selection. Breeders frequently encounter the problem of sterility in either the parental crosses or the subsequent crosses of the progenies (Chuang et al., 2008). One of the major causes of sterility is the difference in chromosome sizes among species and varieties (Arends, 1970). The other cause is the different ploidy levels of the parents, because the commercial varieties/hybrids are usually tetraploid while the wild species are diploid (Greisbach, 1985; Chuang et al., 2008). In order to have an efficient breeding program, there is a need for the information of the genome sizes and ploidy levels of the breeding materials, including the wild species and the parental stocks of the Phalaenopsis orchids. The above information can be obtained by using flow cytometry (Lin et al., 2001; Chen et al., 2013; Chen et al., 2014).

The determination of nuclear DNA content by flow cytometry is useful in the study of the genome size of plants (Doležel and Bartoš, 2005). The C value refers to the DNA amount of the unreplicated haploid genome and is usually constant among individuals of the same species (Bennett and Leitch, 1995). In the study of nuclear DNA content by flow cytometry, the use of intercalating dyes such as propidium iodide (PI) is recommended because they are independent of DNA base composition (Doležel et al., 1992, 1998). On the other hand, other DNA fluorochromes, such as 4, 6-diamidino-2-phenylindole (DAPI) may also be used to measure the relative nuclear DNA contents of species. However, it may over- or underestimate the nuclear DNA content because they bind preferentially to adenine– thymine (AT)–rich regions (Manzini et al., 1983, Doležel et al., 1992). Additional use of this fluorochrome showing base preference may give further insight into the relationship of plant species at the molecular level.

This chapter presents the recent studies on the estimation of the genome size in species of Phalaenopsis using two DNA fluorochromes, PI and DAPI. Examples of the DNA histograms in four species with different chromosome sizes (Kao et al., 2001) are shown in Fig. 1.1. Based on these studies, the relationship between genome size and plant classification, and the application of this information for the improvement of new varieties, are discussed.

1.2.Variation of the Nuclear DNA Content in Phalaenospsis Species Using the PI Fluorochrome

In this study, flowers of 50 Phalaenopsis species were collected from the germplasm banks of several sources in Taiwan during the flowering season (Chen et al., 2013). Since the development of ovules in the ovary of Phalaenopsis is pollination-dependent (Zhang and O’Niell, 1993), the ovary remains as immature somatic tissue with 2C DNA content before pollination. Therefore, sections of the tissue of the young ovary were employed for the analysis by a flow cytometer (Epics XL-MCL, Beckman Coulter, U.S.A.) using PI as the DNA fluorochrome (Chen et al., 2013). Samples of young leaves from the in vitro plantlets of diploid P. aphrodite with a 2C nuclear DNA content of 2.80 picograms (pg) (Lin et al., 2001) were used as standard reference in the analysis by flow cytometry. Values for the fluorescence ratio and the nuclear DNA content of the test sample (i.e. the sample 2C value) were estimated by the following formulae (Doležel and Bartoš, 2005):

Fig. 1.1. DNA histograms of PI-stained nuclei (A–D) and of DAPI-stained nuclei (E–H) of in vitro leaf tissue of reference, P. aphrodite (A, E), and of ovary tissue of P. cornu-cervi (B, F), P. venosa (C, G) and P. violacea (D, H).

Fluorescence ratio = (sample G₀/G₁ peak mean)

(reference G₀/G₁ peak mean);

Sample 2C value = PI fluorescence ratio × 2.80 (pg),

By using this procedure, estimations of the PI-stained fluorescence ratios and the 2C values of the 50 Phalaenopsis species, belonging to two subgenera (Aphyllae and Parishianae) and 8 sections of the subgenera Polychilos and Phalaenopsis (Christenson, 2001), were made; see Table 1.1 (Chen et al., 2013). The variation in the 2C values of the 50 species was high, with a range from 2.77 pg in P. philippinensis to 17.47 pg in P. lobbii. Significant difference was observed in the 2C values among species within each taxonomic subgenus/section with the exception of the section Fuscatae of the subgenus Polychilos and the section Phalaenopsis of the subgenus Phalaenopsis. The means and ranges of the 2C values of 10 taxonomic categories are shown in Table 1.2. The subgenera Parishianae and Aphyllae and two sections of the subgenus Phalaenopsis, namely Esmeralda and Deliciosae, showed large genome sizes with mean 2C values of 15.84, 10.92, 12.29, and 9.63 pg, respectively. The mean 2C values of the sections Phalaenopsis (2.82 pg) and Stauroglottis (3.00 pg) were the lowest among all taxa studied. Furthermore, the range of the 2C values of 19 species of the section Amboinenses varied from 4.53 pg in P. modesta to 13.04 pg in P. bellina. This was the widest range among all taxonomic categories. It was shown that the amounts of the DNA content in 8 Phalaenopsis species were positively correlated with the chromosome size (Kao et al., 2001). Figure 1.2 shows the scatter diagram of 2C values estimated in this study with the total complement lengths of the chromosomes of these 8 species. The linear regression line is y = 7.02x + 27.63 with R² = 0.95, which provides direct evidence for the relationship between the chromosome size and the DNA content measurement.

Table 1.1. The Means of the PI and DAPI Fluorescence Ratios and 2C Values of Phalaenopsis Species

*Species are arranged by the order of the mean 2C values of taxonomic categories and those within each of the category.
^†Number of measurements.
^‡Fluorescence ratio = sample G1 peak mean / standard (diploid P. aphrodite) G1 peak mean.
^§2C values of 50 species measured by PI were calibrated by using diploid P. aphrodite (2C value = 2.80 pg) as the standard.
^||The Mean value for a particular species followed by the same letters within a column is not significantly different at α = 0.05 (Tukey–Kramer′s test).

Table 1.2. The Number of Diploid Species, Mean and Range of 2C Values Within Each Phalaenopsis Subgenus/Section

*The mean value for a particular taxon followed by the same letters within a column is not significantly different at α = 0.05 (Tukey–Kramer′s test).

1.3.Comparison of the Variations of the Fluorescence Ratios in Phalaenopsis Species Using the DAPI and PI Fluorochromes

Flowers of 52 Phalaenopsis species were used to estimate the fluorescence ratio by means of the DAPI fluorochrome in this study. Forty-nine of these species had also been used in the previous stud...

Table of contents

• Cover
• Halftitle
• Title
• Copyright
• Foreword
• Preface
• Contents
• Chapter 1 Genome Size Variation in Species of the Genus Phalaenopsis Blume (Orchidaceae) and Its Application in Variety Improvement
• Chapter 2 Distinct Distribution Patterns of 45S rDNA and 5S rDNA-NTS–Related Repeats Display Diverse Karyotypes in Paphiopedilum
• Chapter 3 Chromosome Number Variation in the Emergent Orchid Model Species Erycina pusilla
• Chapter 4 Comparative Chloroplast DNA Analysis of Phalaenopsis Orchids and Evaluation of cpDNA Markers for Distinguishing Moth Orchids 61 Ching-Chun Chang, Wen-Luan Wu, Yu-Chang Liu,
• Chapter 5 Development of SSR Markers in Phalaenopsis Orchids, Their Characterization, Cross-Transferability and Application for Identification
• Chapter 6 Warm-Night Temperature Delays Spike Emergence and Alters Carbon Pool Metabolism in the Stem and Leaves of Phalaenopsis aphrodite
• Chapter 7 Light-Emitting Diodes Affect the Growth and Gene Expression of Phalaenopsis Plantlets In Vitro
• Chapter 8 Handling, Packaging, and Transportation of Phalaenopsis Plants for Marine Shipment
• Chapter 9 The Orchid-Infecting Viruses Found in the 21st Century
• Chapter 10 Phalaenopsis Viruses and Their Detection Techniques
• Chapter 11 Virus Resistance in Orchids
• Chapter 12 Perianth Regulation in Orchids
• Chapter 13 Flower Development of Phalaenopsis Orchids Involves Functionally Divergent B-Class and E-Class MADS-Box Genes
• Chapter 14 The Function of C/D-Class MADS Box Genes in Orchid Gynostemium and Ovule Development
• Chapter 15 The Dual Function of Harpin in Disease Resistance and Growth in Phalaenopsis Orchids
• Chapter 16 Development of a High-Throughput Virus-Induced Gene-Silencing Vector in Phenotypic Screening of Orchids
• Chapter 17 Application of VIGS to Floral Gene Function Studies of the Orchid, a Nonmodel Plant
• Chapter 18 Functional Study of Phytoene Synthase by RNAi-Based Downregulation in the Oncidesa Orchid
• Chapter 19 Flower Color and Pigmentation Patterns in Phalaenopsis Orchids
• Chapter 20 Prolonged Exposure to Elevated Temperature Induces Floral Transition via Upregulation of Cytosolic Ascorbate Peroxidase 1 and Subsequent Reduction of the Ascorbate Redox Ratio in the Oncidium Hybrid Orchid 421 Dan-Chu Chin and Kai-Wun Yeh
• Chapter 21 Methylation Effect on Chalcone Synthase Gene Expression Determines Anthocyanin Pigmentation in Floral Tissues of Two Oncidium Orchid Cultivars
• Index