Orchid Biotechnology

The following sections are included:

• Foreword

• Preface

• Contents

• List of Contributors

One of the most important strategies to keep Taiwan as the leading producer of Phalaenopsis in the world, is breeding and development of new varieties. Pedigree analysis of the 12 most popular white hybrids of Phalaenopsis indicated that the tetraploids of Phal. amabilis and the hybrid, Phal. Doris were used frequently as parents of these hybrids. Besides the standard big flower Phalaenopsis, development of novelty varieties, such as the Harlequins and the multi-floral types constitute the new trends in the Phalaenopsis breeding programs and markets in the last decade. The somaclonal mutants of Phal. Golden Peoker and the wild species, Phal. equestris played an important role in the development of these novelty varieties. Breeding for new varieties of Phalaenopsis is lengthy and time consuming. New techniques are needed to increase the breeding efficiency of crops having long life cycles. The recent development of molecular markers, such as restricted fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and DNA amplification fingerprinting (DAF) and their applications in Phalaenopsis breeding are discussed and evaluated in this chapter.

The pattern of orchid embryo development is unique among flowering plants. The minute size of the embryos, lack of cotyledon, absence of an endosperm, the varied suspensor morphology, and the simple seed coat structure are some of the unique features of orchid seeds. This chapter summarizes the recent observations on the structural and physiological aspects of orchid embryo development using a few case histories, i.e. Cypripedium formosanum, Calanthe tricarinata and Phalaenopsis amabilis var. formosa. The unique features of orchid embryo development such as: 1) the lack of a clear histodifferentiation pattern; 2) presence of a cuticle over the embryo proper; 3) absence of a cuticle in the suspensor cell wall; 4) a suspensor having a transfer cell morphology; and 5) accumulation of high levels of ABA in mature seeds of some terrestrial species are discussed. A comprehensive understanding of the structural and physiological changes during the orchid embryo development will facilitate successful micropropagation of orchids.

Plant biotechnology, especially in vitro regeneration and flowering, cell biology, DNA manipulation, and biochemical engineering, is reshaping orchid research in four major areas: 1) benefiting micro-propagation and transgenic research with findings on the totipotency and regeneration ability through shoot-bud formation and somatic embryogenesis from callus, direct somatic embryogenesis from explants and thin-section cultures of leaves and roots, and even shoot-bud formation of suspension cells of several major commercial orchids; 2) active research into the dissection of genes responsible for controlling growth, meristem functioning, and flowering of orchids; 3) successful application of molecular genetics and plant transformation under laboratory conditions for protecting commercial orchids against biotic stress; 4) production of specialty biochemicals and pharmaceuticals. These are all good starts, but more devotion and support are needed for further research into both basic and practical aspects. This chapter will include findings on: 1) direct somatic embryogenesis of leaf explants of Oncidium, Phalaenopsis, and Dendrobium; 2) direct shoot-bud formation from leaf explants and regeneration from suspension cells of Paphiopedilum; 3) in vitro flowering of callus-derived somatic embryos and plantlets of Cymbidium, Dendrobium, and Phalaenopsis; and 4) disease-resistant Dendrobium and Oncidium through genetic transformation. For the challenges ahead in orchid biotechnology and its practical application, I strongly encourage integrating modern technologies with classical breeding for the future success of the commercial orchid industry.

Mass production of orchids is achieved through micropropagation of axillary buds of flower stalks or shoot meristem culture. Somaclonal variation occurs during proliferation of both shoots and protocorm-like bodies (PLBs) and leads to morphological or physiological changes in the finished potted plants. We observed both vegetative and reproductive variants, depending on the cultivar or genetic background, among tissue-cultured orchid plants. The use of molecular approaches such as random amplified polymorphic DNA (RAPD), cDNA-RAPD and cDNA suppression subtractive hybridization has resulted in the successful identification of expressed sequence tags (ESTs) from wild-type and peloric flower buds of several Phalaenopsis and Doritaenopsis hybrids. Several candidate genes were cloned and transcript levels compared in these plants. The results suggested that some genes, such as a retroelement, could have been abnormally activated in the peloric mutants. We discuss other investigations, such as DNA methylation status, which might also play a role in somaclonal variations in orchids.

Wild-grown and greenhouse-cultivated orchid roots and the associated fungi were isolated on 1/6 PDA. The fungi isolated from various Taiwan orchid roots were Acremonium spp.; Alternaria spp.; Cylindrocarpon spp.; Fusarium spp.; mycelia sterile; Penecillum spp.; Rhizoctonia spp.; and Trichoderma spp. Those isolates that showed no patho-genecity to the major crops of Taiwan: mungbean, cucumber, radish and rice seedlings were used for inoculation of orchid mycorrhizal fungi. Only those proving beneficial in the growth of orchids were considered as orchid mycorrhizal fungi (OMF). Trichoderma and Rhizoctonia were the most frequent fungi isolates from the Taiwan wild-grown orchid roots. Based on 10 years of inoculation tests, R01, R02, and R04 are shown to be the three isolates with OMF of commercial value. Among them, R01 and R02 are binucleate, while R04 is a multinucleate Rhizoctonia sp. Up till now the identification of R01 and R02 has been uncertain. However, it is known that R04 is Rhizoctonia solani, in the AG6, which has no pathogenic effects on orchids. Seed germinations were enhanced by R02 for Anoectochilus formosanus Hayata and R01 for Haemaria discolor var. dawsoniana. Growth of seedlings was also highly enhanced by the inoculation of Rhizoctonia OMF isolates for three medicinal uses of orchids, such as A. formosanus (R02, R04), H. discolor (R01), and Dendrobium spp. Higher acid and alkaline phosphatases and superoxide dismutase activities, together with flavonoids, phenolic compounds, polysaccharides, ascorbic acids, and phosphate contents were all higher in mycorrhizal tissues than the non-mycorrhizal control of A. formosanus. Vegetative and reproductive growth was stimulated by OMF for ornamental orchids, such as Doritaenopsis spp. (R02, R01, R04), Phalaenopsis spp. (R01, R02, R04), and Phaphiopedium delenatii (R02, R04). Oat meal agar should replace MS or Kyoto agar medium if OMF is inoculated in vitro. The physiological effects of OMF and their potential applications on commercially cultivated orchids are presented. It was highly recommended that the OMF be applied as follows: 1) to increase the survival rates of micropropagated plantlets or seedlings ex vitro; 2) to enhance both vegetative and reproductive growth of orchid plants; 3) to result in earlier flowering and enhanced flower quality; and 4) to reduce disease infection rates.

Flow cytometry is an efficient and reliable method for the estimation of nuclear DNA contents. Nuclei suspension from orchid plant is often contaminated with a high level of crystalline calcium oxalate that blocks the fluidics system of the flow cytometer. A simple and highly efficient protocol enables isolation of intact nuclei from recalcitrant plant tissues containing high levels of polysaccharides, calcium oxalate crystals, and other metabolites. This method was applied to determine the genome sizes of Phalaenopsis sp. using Pisum sativum Minerva Maple as the standard. The occurrence of endoreduplication in orchids during organ development and the influences of environmental perturbations are also discussed.

All Phalaenopsis species have the same chromosome number (2n = 2x = 38), but their karyotypes and genome sizes vary markedly. The variation is positively correlated with the amount of constitutive heterochromatin in the genome. Genomic in situ and Southern hybridizations indicate that species with large genomes contain more repetitive sequences; however, species with small genomes also have their own specific repetitive sequences. One family of tandem repeats named Pvr I consisting of 7-bp repeat units was isolated and characterized. Chromosomal locations of these repeats coincide with heterochromatic blocks.

The complete nucleotide sequence of the chloroplast genome of the Taiwan moth orchid (Phalaenopsis aphrodite subsp. formosana) was determined. The circular, double-stranded DNA of 148,964 bp comprises a pair of inverted repeats of 25,732 bp, which are separated by a small single copy (SSC) and a large single copy (LSC) region of 11,543 and 85,957 bp, respectively. The genome contains 76 protein coding genes, four ribosomal RNA genes, 30 tRNA genes, and 24 putative open reading frames (ORFs). Seventeen genes are intron-containing, including 6 tRNA and 11 protein coding genes. Unlike other chloroplast genomes of photo-synthetic angiosperms, which have a complete set of genes in the 11 sub-units of NADH dehydrogenase, the chloroplast genome of Phalaenopsis completely lacks the ndhA, ndhH, and ndhF genes. The other eight ndh genes have various degrees of nucleotide insertion/deletion, and they are all frameshifted. This loss results in the SSC region in Phalaenopsis being the shortest among known photosynthetic angiosperms.

Orchids have great diversity of specialized pollination and ecological strategies and provide a rich setting for studying evolutionary relationships and molecular biology. The sophisticated orchid flower morphology offers an opportunity to discover new variant genes and different levels of complexity in the morphogenesis of flowers. To obtain plentiful gene information from orchid reproductive organs, we constructed a cDNA library of mature flower buds of Phalaenopsis equestris, a native diploid species of Phalaenopsis in Taiwan. A total of 5,593 expressed sequence tags (ESTs) from randomly selected clones were identified and characterized. Cluster analysis enabled the identification of a unigene set of 3,688 sequences. The abundance of transcripts with predicted cellular roles were functionally characterized by using the BLASTX matches to known proteins. Comparison of the relative EST frequencies based on functional categories among floral tissues of five species including P. equestris, Acorus Americanus, Asparagus officinalis, Oryza sativa, and Arabidopsis thaliana was performed. The most highly transcribed genes in Phalaenopsis floral buds are those coding for RNA-dependent RNA polymerase of Cymbidium mosaic virus, followed by heat shock protein genes. A total 217 putative transcription factor related ESTs were identified. C3H and trihelix families occupied 25% of the transcribed transcription factor genes, indicating that the profile of the transcription factors in orchid flower buds is polarized. The extensive analysis of the genes in floral organs adds to the growing repertoire of known plant genes and may also reveal unique features of the reproductive organs of orchids.

Orchids are known for both their floral diversity and ecological strategies. The versatility and specialization in orchid floral morphology, structure, and physiological properties have fascinated botanists for centuries. In floral studies, MADS-box genes contributing to the now-famous “ABCDE model” of floral organ identity control have dominated conceptual thinking. The sophisticated orchid floral organization offers an opportunity to discover new variant genes and levels of complexity different from the ABCDE model. Recently, several remarkable researches involving orchid MADS-box genes have revealed the important roles of these genes in orchid floral development. Knowledge about MADS-box genes encoding ABCDE functions in orchids will provide insights into the highly evolved floral morphogenetic networks of orchids.

Oncidium pseudobulb is a critical organ in the developmental stage. It is a sink for nutrition, water, and mineral storage organ during the vegetative growth stage. The growth and development determine Oncidium plant growth cycle transforming from the vegetative into the reproductive stage. Therefore, the genes activating in the pseudobulb of before-inflorescence stage attract much research interest. In this chapter, we present a subtractive EST data bank of EST genes, which are actively expressing in the pseudobulb tissues before inflorescence initiation. In total, 74.8% of 636 unique gene parts were annotated on the database of the NCBI GenBank. Largely, all EST was classified into carbohydrate metabolism involved in mannan, pectin, and starch bosynthesis, transportation, stress-related, and regulatory function. Most of the genes that were differentially expressed are involved in early flowering development, carbohydrate metabolism, and stress-response physiology. The efficient pseudobulb-specific EST-library represented an explicit tran-scriptome profile before the flowering stage. It is a valuable data bank for molecular biology study in orchid.

The largest family of angiosperms, Orchidaceae, has diverse, specialized pollination and ecological strategies and provides a rich subject for investigating evolutionary relationships and developmental biology. However, the study of these non-model organisms may be hindered by challenges, such as their large genome size, low transformation efficiency, long regeneration time, and long life-cycle. To overcome these obstacles, we first developed vectors with the use of a symptomless Cymbidium mosaic virus, which infects most orchids, then combined simple physiological controls and virus-induced gene silencing (VIGS) for validation of gene function in orchids. The success of our strategies was verified by our functional validation of floral identity gene(s) in the tetraploid Phalaenopsis orchids, which have an unusually long life-cycle (two years from sowing to flowering). We could knock down the RNA level of either a specific Phalaenopsis floral identity gene or a family of genes congruently in two months. Functional analysis of orchid genes could become easier and profit from the VIGS approach.

Orchids are primarily grown for their large, long-lasting, and fascinating flowers; thus, the improvement of quality attributes such as flower color, longevity, shape, architecture, biotic and abiotic stress tolerance, and creation of novel variations are important economic goals for floriculturists across the world. Recent advances in genetic engineering and molecular biology techniques augmented with gene transformation could help growers to meet the demand of the orchid industry in the new century. Presented is an overview of Agrobacterium-mediated and particle bombardment or direct gene transformation in orchids and the essential factors involved in the technology systems. Available methods for the transfer of genes could greatly simplify traditional breeding procedures and overcome some of the inherent genetic problems, which otherwise would not be achievable through conventional methods. Indeed, more recently, orchids have been the subject of new areas of research, including functional genomics, proteomics, and metabolomics. The successful application of these new approaches to improve traits requires a reliable and reproducible transformation technique. The development and remarkable achievements of biotechnology in orchids during the past decade are reviewed, as are potential areas of research for the improvement of orchids.

The following sections are included:

• Index