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The orchid market is a dynamic horticultural business in which novelty and beauty command high prices. The development of miniature to large and showy flowers, in addition to fragrance, is mainly of interest. Overall organ size might be modified by doubling the chromosome number, which can be accomplished by careful study of meiotic chromosome disjunction in hybrids or species. Meiosis is the process in which diploid (2n) pollen mother cells recombine their DNA sequences and then undergo two rounds of division to give rise to four haploid (n) cells called sporads. Thus, by interfering in chromosome segregation, one can induce the development of diploid recombinant sporads called dyads. These dyads may be used for breeding polyploid progenies with enhanced fertility and large flower size. This review gives an overview of developments in orchid breeding placed in the large context of ploidy breeding in higher plants to facilitate innovation. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL) UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomía

Sex form is one of the most important characteristics in papaya cultivation in which hermaphrodite is the preferable form. Self-pollination of H*-TSS No.7, an inbred line derived from a rare X chromosome mutant SR*, produced all-hermaphrodite progeny. The recessive lethal allele controlling the all-hermaphrodite phenomenon was proposed to be the recessive Germination suppressor (gs) locus. This study employed next-generation sequencing technology and genome comparison to identify the candidate Gs gene. One specific gene, monodehydroascorbate reductase 4 (MDAR4) harboring a unique polymorphic 3 bp deletion in H*-TSS No.7 was identified. The function of MDAR4 is known to be involved in the hydrogen peroxide (H2O2) scavenging pathway and is associated with seed germination. Furthermore, MDAR4 showed higher expression in the imbibed seeds than that in the dry seeds indicating its potential role in the seed germination. Perhaps this is the very first report providing the evidences that MDAR4 is the candidate of Gs locus in H*-TSS No.7. In addition, Gs allele-specific markers were developed which would be facilitated for breeding all-hermaphrodite lines.

The orchid market is a dynamic horticultural business in which novelty and beauty command high prices. The two main interests are the development of flowers, from the miniature to the large and showy, and their fragrance. Overall organ size might be modified by doubling the chromosome number, which can be accomplished by careful study of meiotic chromosome disjunction in hybrids or species. Meiosis is the process in which diploid (2n) pollen mother cells recombine their DNA sequences and then undergo two rounds of division to give rise to four haploid (n) cells. Thus, by interfering in chromosome segregation, one can induce the development of diploid recombinant cells, called unreduced gametes. These unreduced gametes may be used for breeding polyploid progenies with enhanced fertility and large flower size. This review provides an overview of developments in orchid polyploidy breeding placed in the large context of meiotic chromosome segregation in the model plants Arabidopsis thaliana and Brassica napus to facilitate molecular translational research and horticultural innovation. Universidad de Costa Rica/[814-C2-060]/UCR/Costa Rica Universidad de Costa Rica/[814-C0-244]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL) UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomía

Orchidaceae is one of the largest families of flowering plants with about 30,000 species. Orchid flowers are highly valued because of their quite distinct and pretty features like spectacular floral shape, color, and elaborate adaptations to pollinators. Among the numerous species, the Phalaenopsis genus of about 66 species derived hybrids is the popular potted ornamental plant. Nowadays, there are more commercial demands for the various Phalaenopsis hybrids with diverse flower colors, fragrance, disease-free, environmentally protected with long-lasting blooming. To produce Phalaenopsis hybrids with diverse features, the whole-genome organization analysis plays a key role to deploy suitable strategies. Genome sequencing has been developed as an important discipline in the plant sciences. During the past decade, remarkable advancement has been made in next-generation sequencing technologies, including 454 pyrosequencing, Illumina/Solexa technology, and sequencing by oligo ligation detection. To accelerate genetic improvement through molecular breeding, wide-ranging analyses of genes and the genome of Phalaenopsis have been conducted using advanced technologies. Whole-genome and transcriptomes were sequenced, assembled, and annotated in (wild-type and peloric mutant plants) P. equestris, P. aphrodite and hybrids. The chloroplast DNA sequence information of P. equestris and P. aphrodite were obtained. Various RNA-Seq reads were produced from flowers and temperature treated parts of Phalaenopsis orchids (wild-type and peloric mutant plants) and P. bellina. From these produced genome sequences, various databases were constructed for the Phalaenopsis genome. We expect Phalaenopsis genome sequences, microRNAs, expressed sequence tags, simple sequence repeats, linkage maps, and constructed databases will deliver a valued source for basic and applied research, including comparative genomic analysis, developmental studies and molecular breeding on Phalaenopsis.

Orchids have flowers of unique beauty that are remarkable for their zygomorphic syndrome, which can be summarized as a floral architecture based on three categories of organs at the perianth: external tepals, internal lateral tepals, and a labellum or lip, a prominent central inner petal believed to be a specialized adaptation that attracts pollinators. These mesmerizing floral traits have enthralled researchers into the study of the orchid homologs of the MADS-box family of genes, which are transcriptional factors believed to be spatiotemporal determinants of organ identity during floral development. The identification of several putative members of the MADS-box family may clarify their potential role in orchid flower development, especially during the transition to flowering and during the patterning of orchid flower organs. Furthermore, we look into new technologies of genome analysis and gene editing in order to appraise potential applications for basic research purposes and for the breeding of new orchid varieties. Universidad Nacional de Ciencia y Tecnología de Pingtung/[]/NPUST/Taiwán Universidad de Costa Rica/[]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL)

The global orchid production has been active in tropical, subtropical countries, many with automated and efficiently managed large greenhouses. The most important exported potted orchid in the trade is Phalaenopsis with tremendous breeding and micropropagation technology achievements in several countries, such as Belgium, the Netherlands, Taiwan, and Thailand. Another reason for its popularity is its easiness of controlled flower induction for scheduled and year-around production. Cut orchids such as Cymbidium, Dendrobium, Oncidium, and Vanda are important cut flowers worldwide. Now orchids are quite popular in the global market, partly due to their long shelf life, diverse colors, and other desirable traits pursued by consumers, depending on the location or culture. European Union (EU) and the United States are the most important countries or areas for marketing of assortments of orchid products. New markets for orchids are increasing annually. Since the beginning of 2020, due to the severe coronavirus pandemic and disruption of global economy, orchid marketing has been shifting to an increased share of digitalization or E-commerce, co-existing with the traditional flower stores and supermarkets. In the future, more orchid products or novel new orchid hybrids being developed by non-conventional technologies, such as biotech-oriented efficient breeding, may help offer new opportunities for orchid production.

Erycina pusilla is an orchid species from the tropics with a wide distribution in neotropical America. It is a fast-growing tiny twig epiphytic orchid and blooms constantly throughout the year. Approximately 28 MIKC/MADS-box genes from E. pusilla have been identified.Type-II MADS-box proteins (also called MIKC-domain proteins) play a crucial role during flower development, making this species an ideal orchid model for developmental studies. E. pusilla has a small diploid genome and two different alternative cytotypes have been reported, with 10 and 12 chromosomes. The karyotype evolution in Erycina is the result of processes of segment translocation and heterochromatin expansion/deletion. In order to breed new commercial orchid species, E. pusilla has been crossed with several important Oncidiinae orchids. The clone PSYP1 as E. pusilla ‘Hsingda Golden’ derived from in vitro flowering system has been granted the Plant Variety Rights in Taiwan for protection. The E. pusilla plants can also be engineered using the Agrobacterium tumefaciens strain EHA105 together with a plasmid vector carrying the CaMV 35S promoter system. The twig epiphyte E. pusilla holds enormous biotechnological and horticultural potential. It may be necessary to attempt more basic and applied research to transform the current knowledge into new Oncidium varieties with altered flower morphology, enhanced growth rate and vibrant colors. Universidad Nacional de Ciencia y Tecnología de Pingtung/[]/ NPUST/Taiwán Universidad de Costa Rica/[]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL)

The lush and exuberant subtropical forests of South East Asia are home to about 60 native species of Phalaenopsis orchids. Results from research into evolutionary and reproductive relationships within this genus are reported with the hope that their understanding may assist the selection of species as parents during breeding programs. The species in sub-genus Phalaenopsis have been used frequently for commercial breeding and trading. Polyploid cultivars with superior horticultural traits are the mainstream, and are the product of either the natural production of unreduced gametes or the result of artificial production during the process of cross hybridization. Meiocyte analysis may provide valuable information regarding the formation of unreduced gametes and subsequent use in breeding for polyploidization. Artificial induction of polyploidy may be brought about by chemical treatments or as an unintended result of mutations during the tissue culture process. Proper storage of orchid pollinia in sub-zero freezing temperatures may extend their viability until the moment they are required for cross hybridization. This chapter elaborates into these methods and strategies with the hope that they might contribute to new orchid breeding programs. National Pingtung University of Science and Technology/[]/NPUST/Taiwan Universidad de Costa Rica/[]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL)

Orchids constitute the largest families within the flowering plants; they are one of the most highly evolved groups in the angiosperms; and because of their flashy flowers many species from several genera are very prominent horticultural commodities. Environmental factors such as temperature and daylength influence flowering; however, other factors play a major role such as the developmental stage, plant hormonal regulation and genetic factors. Research on model plants such as the thale cress (Arabidopsis thaliana) and rice (Oryza sativa) has allowed a broad understanding of the genetics and the molecular regulation of flowering in most plants. However, until very recently the specifics of floral regulation in orchids had remained quite obscure. In this review we have examined very recent work concerning flowering of orchids with a focus on the interplay between development, environmental cues, plant hormones, and gene networks. We have highlighted several examples of successful manipulation of flowering through biotechnology that may be very relevant for researchers and growers elsewhere. Universidad Nacional de Ciencia y Tecnología de Pingtung/[]/NPUST/Taiwán Universidad de Costa Rica/[]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM) UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Jardín Botánico Lankester (JBL)

This chapter provides information on the floral development and flowering, sex determination and their molecular markers in pawpaws (Carica papaya). Floral morphology, environmental factors, plant nutrition, gene regulation and plant growth regulators affecting floral development and sex expression are also discussed.

Cut foliage of Monstera deliciosa is widely used in flower arrangement due to its long vase life and striking foliage pattern. Variegated Monstera of commercial importance is scarce. Therefore, we examined the potential of mutagenesis in obtaining variegated mutants by gamma ray irradiating the seeds. The germination rate was reduced by increasing the irradiation dosage. The LD50 dosage was between 10 to 25 Gy. We further compared the survival rate of seeds from different source at 0, 7.5, 10, and 12.5 Gy. Dosage higher than 12.5 Gy was required to achieve LD50 germination rate. Next, seeds pre-soaked for 5 days were compared to dry seeds for their sensitivity to gamma irradiation at various dosages (10, 12.5, 15, 17.5, 20, and 22.5 Gy). The highest germination rate was obtained by 10 Gy of dry seeds and 12.5 Gy of soaked seeds. The lowest germination rate was observed by 22.5 Gy for both dry and soaked seeds. The soaked seeds apparently were more tolerant to the damage caused by higher dosage of irradiation. Different pattern of leaf variegation was obtained after gamma ray irradiation, including light green to yellow green sectors.

Orchids comprise one of the largest, most highly evolved angiosperm families, and form an extremely peculiar group of plants. Various orchids are available through traditional breeding and micro-propagation since they are valuable as potted plants and/or cut flowers in horticultural markets. The flowering of orchids is generally influenced by environmental signals such as temperature and endogenous developmental programs controlled by genetic factors as is usual in many flowering plant species. The process of floral transition is connected to the flower developmental programs that include floral meristem maintenance and floral organ specification. Thanks to advances in molecular and genetic technologies, the understanding of the molecular mechanisms underlying orchid floral transition and flower developmental processes have been widened, especially in several commercially important orchids such as Phalaenopsis, Dendrobium and Oncidium. In this review, we consolidate recent progress in research on the floral transition and flower development of orchids emphasizing representative genes and genetic networks, and also introduce a few successful cases of manipulation of orchid flowering/flower development through the application of molecular breeding or biotechnology tools.

The study of genetic diversity in different Jatropha curcas L. accessions cultivated in Taiwan comprises a necessary first step in germplasm evaluation towards improving this plant species. Genetic variations of 78 Jatropha curcas L. accessions representing origin of eight countries, cultivated in two locations in Taiwan were determined using inter simple sequence repeats (ISSR) markers. Out of 22 screened UBC primers set No. 9, ten primers amplified a total of 27 loci with 23 be polymorphic (85.19 %). Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis indicated two main clusters with the Dice coefficient of similarity ranging from 0.62 to 1.00. Although the grouping was not related to cultivation locations, there was close genetic relationship among the Jatropha populations. Analysis of molecular variance revealed 31.23 % of the variability attributable to among populations with the remaining 68.77 % within Jatropha populations. The overall analysis of different populations suggests less variation of cultivated Jatropha populations in Taiwan.

The papaya (Carica papaya L.) is one of the most important economic tropical fruits in the world, and the hermaphrodite is the preferred type in field cultures. We analyzed the sexual ratio of offspring from the cultivar 'Taiwan Seed Station No. 7' (T7) by a self-cross and its cross with Taichung Sunrise (TS). Female progeny from the T7 self-crossing were not observed. This finding may be caused by a lethal gene that is linked to females. In this study, we selected 192 simple sequence repeats (SSRs) to analyze the polymorphism between T7 and TS. A total of 37 SSRs were identified for T7 and TS. In addition, 14 SSRs served as the molecular makers for identification of T7, TS and their hybrid offsprings. Thus, the results show that the genetic similarity between T7 and TS is rather high. This suggests that T7 may be a mutant of TS. Phylogenetic analysis from the SSR polymorphisms of the above parent strains and 15 F1 offspring revealed the genetic distance of the F1 offspring located between T7 and TS. The results of this study may provide an opportunity for elucidating the genetic characteristics of all hermaphrodites via identification of molecular makers.

Rice (Oryza sativa L.) is one of the most important food crops in the world. In Taiwan, due to the warm climate, there are two harvests annually. However, the yield and quality of rice can vary between each crop season in any given year. Previous reports have shown that microRNAs (miRNAs) play a crucial role in many developmental and physiological processes in plants. In this study, the heading date characteristics of 167 rice cultivars from the second crop season were recorded, and 27 rice cultivars were selected for preliminary microarray analysis. A total of 14 miRNAs from different heading date characteristics in 21 cultivars were selected based on significant differences in their expression profiles. Using a correlation analysis between the heading date and selected miRNA expression obtained from real-time polymerase chain reaction (PCR) assays, we developed a heading date prediction model. The model includes nine miRNA genes with corresponding R2 values of 0.8. To confirm the model, a real-time PCR analysis was performed on an additional 27 rice cultivars and we found the model predicted the heading date with accuracy. Therefore, the developed prediction may be useful in further studies aimed at confirming the reliability of the use of miRNA in molecular breeding and to increase the selection efficiency of rice cultivars and breeding.

Carica papaya L. is an important economic crop worldwide and is used as a model plant for sex-determination research. To study the different flower sex types, we screened sex-related genes using alternative splicing sequences (AS-seqs) from a transcriptome database of the three flower sex types, i.e., males, females, and hermaphrodites, established at 28 days before flowering using 15 bacterial artificial chromosomes (BACs) of C. papaya L. After screening, the cDNA regions of the three sex-related loci, including short vegetative phase-like (CpSVPL), the chromatin assembly factor 1 subunit A-like (CpCAF1AL), and the somatic embryogenesis receptor kinase (CpSERK), which contained eight sex-related single-nucleotide polymorphisms (SNPs) from the different sex types of C. papaya L., were genotyped using high-resolution melting (HRM). The three loci were examined regarding the profiles of the third whorl, as described below. CpSVPL, which had one SNP associated with the three sex genotypes, was highly expressed in the male and female sterile flowers (abnormal hermaphrodite flowers) that lacked the fourth whorl structure. CpCAF1AL, which had three SNPs associated with the male genotype, was highly expressed in male and normal hermaphrodite flowers, and had no AS-seqs, whereas it exhibited low expression and an AS-seqs in intron 11 in abnormal hermaphrodite flowers. Conversely, carpellate flowers (abnormal hermaphrodite flowers) showed low expression of CpSVPL and AS-seqs in introns 5, 6, and 7 of CpSERK, which contained four SNPs associated with the female genotype. Specifically, the CpSERK and CpCAF1AL loci exhibited no AS-seq expression in the third whorl of the male and normal hermaphrodite flowers, respectively, and variance in the AS-seq expression of all other types of flowers. Functional mapping of the third whorl of normal hermaphrodites indicated no AS-seq expression in CpSERK, low CpSVPL expression, and, for CpCAF1AL, high expression and no AS-seq expression on XYh-type chromosomes.

Somatic embryogenesis receptor-like kinase (SERK) genes have been found to be involved in the somatic embryogenesis of several plant species. We identified and characterized 5 PhSERK genes in the Phalaenopsis orchid. The amino acid sequences of PhSERKs and other SERK proteins are highly conserved, with the highest homology observed in the leucine-rich repeat-receptor-like kinase domain. All 5 PhSERKs were expressed in all Phalaenopsis organs examined (root, leaf, shoot apical meristem, and flower), with the strongest expression, particularly for PhSERK1 and 3, in the shoot apical meristem of mature plants. Expression of all PhSERKs was downregulated during early floral bud development and was upregulated gradually until the semi-open flower stage was reached. All 5 PhSERKs were expressed during both seed germination and protocorm-like-body (PLB) development. In germinated seeds, quantitative real-time PCR revealed upregulation of all PhSERKs except PhSERK4 at 1 week and downregulation after 4 weeks. The 5 PhSERKs were differentially expressed in the early stage of PLB development and maintained substantial levels during PLB formation, with PhSERK1 and 5 upregulated 1 week after culture and PhSERK2, 3, and 4 downregulated over this period. Because physical wounding of PLB stimulates secondary PLB formation, the PhSERK5 expression peak at week 3 coincided with visible and fully developed secondary PLBs. PhSERK5 may be important in PLB induction and subsequent development. Our PhSERK expression analysis revealed that these genes have a broad role during orchid plant development.

Orchid is a major floral crop around the world and Dendrobium hybrids are considered to be one of the most popular orchids. In vitro germination of hybrid seeds is a common practice among orchid growers, however, in many cross pollinations the embryos may not develop to maturity, leading to poor seed germination. The effect of seed maturity and sucrose concentration were investigated via asymbiotic germination of nobile Dendrobium hybrids. Capsules were harvested from two hybrids (Den. Lucky Girl × Den. Second Love ‘Kirameki’ and Den. Lucky Girl × Den. Hamana Lake ‘Kumi’) and one selfing of Den. Second Love ‘Kirameki’ at 2, 3, 4, and 5 months after pollination and immature seeds were taken. Immature seeds from 3- to 5-month old capsules could be successfully germinated on Hyponex based medium. Immature seeds from 4-month old capsules showed greatest germination rate of tested treatments, whereas 3-month old immature seeds showed the least germination. After 6 weeks of in vitro culture, protocorms derived from embryos developed on every concentration of sucrose, but germination was greater at lower concentrations. Greater concentration of sucrose decreased normal-developed protocorms.

Cut flowers of the orchid Oncidium Gower Ramsey are valuable agricultural produce around the world because of their yellow color contributed by carotenoids in adaxial and abaxial epidermal layers in petals and labellum. Here, we investigated the regulation of carotenogenic genes in response to different light qualities in Oncidium flowers. Total chlorophyll content was high in young flower buds and reduced during flower development, but carotenoid content was increased during flower development. We detected 11 key carotenoid biosynthesis genes and determined their expression profiles with light treatment. Real-time PCR revealed high mRNA levels of most genes, except OncCHYB and OncSDR, in vegetative tissues; only OncLCYe showed low expression in flowers. White light upregulated OncPSY, OncLCYb, and OncNCED but downregulated OncPDS, OncZDS, OncCrtISO, OncLCYe, OncCHYB, OncZEP, OncCCD1, and OncSDR; red light upregulated OncPSY, OncLCYb, OncLCYe, and OncCCD1; and UV light upregulated OncPSY, OncPDS, OncLCYb, OncZEP, and OncNCED. We cloned the promoter region of the phytoene synthase gene and found it to have potential cis-regulatory elements, such as TATA-box and CAAT-box, in addition to multiple light-responsive elements such as G-box, I-box, GT-1 motif, GAG-motif, and H-box. Our results of light-regulated carotenogenic gene expression and promoter analysis in flowers of Oncidium Gower Ramsey suggest ways to manipulate carotenoid content for high-quality cut-flower production.

The popularity of the nobile-type dendrobium (Dendrobium nobile hybrids) has been increasing globally. More information regarding the effects of long-distance shipping, from producing countries to destination market countries, on the post-shipping plant performance is needed. In this study, two nobile-type dendrobium cultivars were subjected to simulated dark shipping (SDS) at various temperatures and durations. Changes in net CO2 uptake rate (Pn), chlorophyll fluorescence, and leaf relative water content after plants had been treated with SDS were investigated. Furthermore, shipped plants were vernalized to investigate the effect of dark shipping on the subsequent flowering quality. Dark shipping for 7 days at 15 °C did not affect the post-shipping photosynthetic performance of D. Lan Tarn Beauty. Increasing the shipping duration from 7 to 21 days increased the time required for Pn recovery from 1 to 12 days. Dendrobium Lan Tarn Beauty recovered its Pn within 4 days when shipped for 21 days at 10 °C, and this was prolonged to 11 days when the plants were shipped at 20 °C. Changes in Fv/Fm indicated that there was no marked damage to either cultivars, and the leaf relative water content was little affected by SDS. Dendrobium Lan Tarn Beauty and D. Lucky Girl shipped at 10 °C flowered 5 and 8 days earlier, respectively, compared with unshipped plants. Regardless of the shipping conditions, shipped D. Lucky Girl had a lower flower diameter and higher total flower count than unshipped plants. No differences were found in the number of nodes with flowers or the total flower count between shipped and unshipped D. Lan Tarn Beauty. Our study suggested that dark shipping for up to 21 days is possible for nobile-type dendrobiums. We recommend shipping temperatures of 10 to 15 °C to reduce the detrimental effects caused by long-term dark shipping.

Interspecific hybrids between cultivars of eggplant (Solanum melongena L.) and its wild relative S. torvum, which has disease resistance and desirable traits for crop improvement, were obtained by cross-hybridization and embryo rescue. Twenty-one hybrid progenies were obtained and examined based on morphological traits, RAPD and ISSR markers. Five of them were confirmed to be true interspecific hybrids. Eighteen and 14 bands from 7 RAPD and 14 ISSR primers, respectively, were polymorphic and present in all five hybrid seedlings and their parents. The morphological characteristics of leaf margin, inflorescence type and spine positions of the five seedlings were intermediate to the parents. These interspecific hybrids had low pollen viability, probably due to abnormal meiosis.

In vitro grown cabbage (Brassica oleracea var. capitata) seedlings exposed to excess molybdenum (Mo) ions exhibited severely reduced plant growth at the cotyledonary stage. Adding 80 mM proline (Pro) to the Mo-treated medium could help 50% seedlings to overcome the toxicity and grow true leaves. Under excess Mo stress, seedlings accumulated blue/purple anthocyanin in their cotyledons and hypocotyls. The anthocyanin content under Mo with 40 mM Pro was 4-fold higher than the control medium, MS with 40 mM Pro. The presence of Pro in the excess-Mo condition reduced chlorophyll a, whereas the chlorophyll b content was much higher than the control media of MS with and without Pro. Moreover, supplementing various concentrations of Pro into the Mo-stressed condition promoted the seedlings with higher antioxidant enzyme activities of superoxide dismutase, ascorbate peroxidate, and catalase. In addition, genes in the anthocyanin biosynthesis and accumulation pathways, phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), flavonone 3-hydroxylase (F3H), leucoanthocyanidin dioxygenase (LDOX), and glutathione-S-transferase (GST), were all upregulated. Our study indicated that, under excess Mo stress, the antioxidant activity of cabbage seedlings was induced in an attempt to protect plants from the Mo-induced toxicity and exacerbated growth. Pro, on the other hand, functioned in producing higher antioxidant enzyme activity to partially help recover plant growth.

ThePhalaenopsisorchid is an important potted flower of high economic value around the world. We report the 3.1 Gb draft genome assembly of an important winter floweringPhalaenopsis‘KHM190’ cultivar. We generated 89.5 Gb RNA-seq and 113 million sRNA-seq reads to use these data to identify 41,153 protein-coding genes and 188 miRNA families. We also generated a draft genome forPhalaenopsis pulcherrima‘B8802,’ a summer flowering species, via resequencing. Comparison of genome data between the twoPhalaenopsiscultivars allowed the identification of 691,532 single-nucleotide polymorphisms. In this study, we reveal that the key role ofPhAGL6bin the regulation of labellum organ development involves alternative splicing in the big lip mutant. Petal or sepal overexpressingPhAGL6bleads to the conversion into a lip-like structure. We also discovered that the gibberellin pathway that regulates the expression of flowering time genes during the reproductive phase change is induced by cool temperature. Our work thus depicted a valuable resource for the flowering control, flower architecture development, and breeding of thePhalaenopsisorchids.

Phalaenopsis orchid is an important potted flower with high economic value around the world. We report the 3.1 Gb draft genome assembly of an important winter flowering Phalaenopsis ‘KHM190’ cultivar. We generated 89.5 Gb RNA-seq and 113 million sRNA-seq reads to use these data to identify 41,153 protein-coding genes and 188 miRNA families. We also generated a draft genome for Phalaenopsis pulcherrima ‘B8802’, a summer flowering species, via resequencing. Comparison of genome data between the two Phalaenopsis cultivars allowed the identification of 691,532 single-nucleotide polymorphisms. In this study, we reveal the key role of PhAGL6b in the regulation of flower organ development involves alternative splicing. We also show gibberellin pathways that regulate the expression of genes control flowering time during the stage in reproductive phase change induced by cool temperature. Our work should contribute a valuable resource for the flowering control, flower architecture development, and breeding of the Phalaenopsis orchids.

The present study describes a direct shoot regeneration-based micropropagation procedure for the self-heading cultivars of Philodendron. Three types of explant (i.e. leaf lamina, petiole, stem nodal segment) were screened for their shoot induction potential following a three months treatment with 0.5 mg l−1 of either 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) or both. Results indicated that the leaf laminas were poor candidates for shoot induction whereas the petioles showed potential for adventitious shoot production at frequencies of 2.8–11.1% in two of the cultivars tested. Stem nodal segments were the most responsive among the three as shoots formed directly following the TDZ treatment at frequencies of 16.7–41.7% depending on the cultivar. When comparing the effectiveness of different cytokinins to induce shoot proliferation on stem nodal segments, it was found that the 0.5 and 1 mg l−1 of kinetin (Kn) and 6-benzyladenine (BA) treatments resulted in higher shoot formation percentages compared to the 0.5 and 1 mg l−1 of TDZ treatments in two of the three cultivars. Furthermore, more shoots were produced on BA than on Kn-supplemented media in all the three cultivars. Shoots derived from the 0.5 mg l−1 of BA treatment can be induced to root following one month incubation with 0.1–1 mg l−1 of indole-3-butyric-acid (IBA). The rooted shoots showed 100% survival after acclimatization in the greenhouse. The procedure reported in the present study can assist in the large-scale multiplication of elite self-heading cultivars of Philodendron in the future.

articulo -- Universidad de Costa Rica. Escuela de Agronomia, 2010. Este documento es privado debido a limitaciones de derechos de autor.

Two pathways are used by higher plants for the biosynthesis of isoprenoid precursors: the mevalonate pathway in the cytosol and a 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids, with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) catalyzing the last step in the MEP pathway. In order to understand the contribution of MEP pathway in isoprenoid biosynthesis of Oncidium orchid, a full-length cDNA corresponding to HDR from the flower tissues of Oncidium Gower Ramsey was cloned. The deduced OncHDR amino acid sequence contains a plastid signal peptide at the N-terminus and four conserved cysteine residues. RT-PCR analysis of HDR in Oncidium flowering plants revealed ubiquitous expression in organs and tissues, with preferential expression in the floral organs. Phylogenetic analysis revealed evolutionary conservation of the encoding HDR protein sequence. The genomic sequence of the HDR in Oncidium is similar to that in Arabidopsis, grape, and rice in structure. Successful complementation by OncHDR of an E. coli hdr(-) mutant confirmed its function. Transgenic tobacco carrying the OncHDR promoter-GUS gene fusion showed expression in most tissues, as well as in reproductive organs, as revealed by histochemical staining. Light induced strong GUS expression driven by the OncHDR promoter in transgenic tobacco seedlings. Taken together, our data suggest a role for OncHDR as a light-activated gene.

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post-transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

Two cultivars of Anthurium andraeanum Hort. hybrids, `Paradise Pink' and `Tropic Flame', were transformed by Agrobacterium to contain gene sequences for Shiva-1, a cecropin-based lytic peptide. The antibacterial gene was driven by a 35-35S cauliflower mosaic viral (35-35S CaMV) promoter and the construct included the secretory signal sequence for pathogenesis-related protein 1b (PR1b). Blight tolerance of regenerated plants was tested by inoculation with a virulent strain of Xanthomonas axonopodis (formerly campestris) pv. dieffenbachiae (Xad) that is bioluminescent to allow detection of symptomless infections in Shiva-1 transformants. Primary regenerants for two Shiva-1 transgenic lines of `Paradise Pink' displayed significantly enhanced tolerance to bacterial blight over blight susceptible `Rudolph' and even the blight tolerant `Kalapana'. Two Shiva-1 transgenic lines of `Tropic Flame' showed no improved resistance when compared to the control at the mean percent leaf infection level. One Shiva-1 transgenic line of `Tropic Flame' was unexpectedly more susceptible to blight than the nontransgenic control. Low expression of Shiva-1 observed in this line is hypothesized to be the cause of its increased susceptibility to Xad.

Papaya (Carica papaya L.) plants are polygamous, with female, male, and hermaphroditic flowers. To understand the roles of MADS-box genes in flower development and sex determination, we cloned cDNAs of E-class genes CpMADS1 and CpMADS3 and a TM6 lineage of the B-class gene CpMADS2 from young flower buds of papaya. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses revealed that CpMADS1 and CpMADS3 were preferentially expressed in the carpel and also in petals and stamens. CpMADS2 was expressed in both petals and stamens early during floral development. Comparison of 10 papaya genotypes of 5 different sex phenotypes - hermaphrodite, male, female, progeny-all-hermaphrodite, and progeny-all-male - by Southern blot analysis of genomic DNAs with probes of the 3 genes revealed similar restriction patterns and copy number, suggesting a low relationship of the 3 CpMADS genes with sex expression of papaya plants at the genomic level.

The Orchidaceae is one of the largest and most diverse families of flowering plants. The Dendrobium genus has high economic potential as ornamental plants and for medicinal purposes. In addition, the species of this genus are able to produce large crops. However, many Dendrobium varieties are very similar in outward appearance, making it difficult to distinguish one species from another. This study demonstrated that the 12 Dendrobium species used in this study may be divided into 2 groups by internal transcribed spacer (ITS) sequence analysis. Red and yellow flowers may also be used to separate these species into 2 main groups. In particular, the deciduous characteristic is associated with the ITS genetic diversity of the A group. Of 53 designed simple sequence repeat (SSR) primer pairs, 7 pairs were polymorphic for polymerase chain reaction products that were amplified from a specific band. The results of this study demonstrate that these 7 SSR primer pairs may potentially be used to identify Dendrobium species and their progeny in future studies.

Thai Agricultural Research Journal, 32, 2, 201-207

Root explants from in vitro-grown plants of Anthurium andraeanum ‘Alii’ and Anthurium interspecific hybrid ‘UH1060’ readily produced multiple shoots under weak light on a modified Murashige & Skoog medium containing 2.2 μM BA. Regenerated ‘UH1060’ plants grew normally and flowered within 16 months after transfer to the greenhouse. Cocultivation of root cuttings with Agrobacterium tumefaciens LBA4404 carrying the binary vector pCa2Att resulted in kanamycin-resistant ‘Anuenue’ shoots transgenic for neo and att and recovered more than one year after culture on selection media. Transformation efficiency (number of explants with transgenic shoots per total explants) was 1.3%. Other cultivars (‘UH1003’, ‘UH1060’, ‘Rudolph’, and ‘Mauna Kea’) failed to produce shoots under the transformation condition employed.

Several procedures were tested in development of a gene transfer protocol for anthurium. Etiolated internode segments of anthurium cultivars 'Rudolph' and 'UH1060' were co-cultured with Agrobacterium tumefaciens LBA4404 carrying the chimeric genes neo, for antibiotic resistance, and att encoding antibacterial attacin. Assays of genomic DNA and RNA from kanamycin-resistant 'Rudolph' and DNA from 'UH1060' plantlets, recovered as soon as 1 year after culture on selection media, indicated the presence of introduced genes, including neo and att, and transcription of att. Western analysis confirmed the expression of attacin protein in calli induced from laminae of regenerated kanamycin- resistant 'Rudolph' plantlets. Use of tobacco nurse cells during co-cultivation of internodes with Agrobacterium did not increase recovery of shoots under the regeneration conditions used. Improvements in culture and antibiotic selection conditions during plant development are suggested. Anthurium hybrids are used worldwide for cut flowers and flowering potted plants. Hawaii produces about one-fifth of the world's export supply with over 10 million stems sold in 1994 (Hawaii Agricultural Statistics Service, 1995). Due to the recent devastation of many Hawaiian anthurium farms by bacterial blight caused by Xanthomonas campestris pv. dieffenbachiae McCull and Pirone, there is a need for blight resistant cultivars. A transfor- mation method to introduce antibacterial genes into this commer- cial monocot is desirable to complement current hybridization breeding for disease resistance (Kamemoto et al., 1992). Such a method could allow introgression of other desirable traits, such as novel flower color or nematode resistance. Feasibility of Agrobacterium -mediated gene transfer has been demonstrated in several monocot crops (Conner and Dommisse, 1992). We showed that etiolated internodes of Anthurium andraeanum Lind. ex Andre 'Rudolph' (formerly UH965) were suitable for infection by wild-type Agrobacteriurn tumefaciens (Smith and Townsend) Corm strain C58; plants were not regener- ated from tumors (Kuehnle and Sugii, 1991a). This report describes several procedures tested in development of a transformation protocol for etiolated anthurium internodes by non-tumorigenic A. tumefaciens LBA4404 using tissue culture and plant regeneration methods for Hawaiian cultivars (Kuehnle and Sugii, 1991b). The first example of transgenic anthurium plants is presented.

Paphiopedilum orchids are generally propagated through the division of axillary buds from mother plant, which limits commercial production due to its unproductive proliferation and time consuming. In this study, the effect of in vitro cutting methods and medium composition on efficient shoot multiplication of Paphiopedilum Hsinying Rubyweb was investigated. Among three different in vitro stem cutting methods used, vertical cutting was able to produce more new shoots than horizontal and cross cutting when cultured on Hyponex based medium. After 12 weeks of culture, plantlets regenerated from vertical cutting were able to produce new healthy and well rooted shoots higher than without cutting on the same medium. Moreover, the newly-formed shoots which were divided into single plantlets and subcultured onto half-strength Murashige and Skoog (MS) medium without growth regulators could remain higher shoot multiplication than in other media. The micropropagation procedure developed in this study provides a simple means to in vitro propagate Paphiopedilum plantlets which are able to produce large numbers of uniform plantlets in a shorter time compared to the conventional propagation method.Key words: Micropropagation, shoot multiplication, cutting, Paphiopedilum.

artículo -- Universidad de Costa Rica. Escuela de Agronomía, 2008. Este documento es privado debido a limitaciones de derechos de autor de la revista. The development of new cultivars in Doritaenopsis Guillaum. & Lami orchids is often hindered by factors such as low seed count in hybrids. Cytological study may offer the ability to develop new hybrids by revealing cultivars with good chromosome pairing and high pollen viability, which are somewhat difficult to obtain under current breeding programs. Cross pollination, pollen viability, and chromosomal behavior during meiosis were analyzed to reveal the relation between seed fertility and capsule set in Doritaenopsis hybrids. The number of mature capsules harvested and their relative seed content were used as indices of crossing availability. The results of meiosis were evaluated according to pollen viability detected by fluorescein diacetate and quantification of sporad types by acid fuchsin staining. Chromosome number and pairing at meiosis were observed in root tips or in samples of pollen mother cells. A positive relation was found among high seed set, high frequency of viable tetrads, high degree of chromosome pairing, and low frequency of chromosomal aberrations such as inversions and translocations. On the basis of these factors, three types of hybrids could be distinguished. In type one hybrids, chromosomes paired as bivalents, pollen mother cells divided into tetrads, and capsule setting occurred after pollination of pollen acceptors. In type two hybrids, chromosomes remained mainly as univalents that developed into micromeiocytes, pollen mother cell division was disrupted, and seed recovery was low after pollination. Type three hybrids showed chromosomes paired mostly as multivalents, chromosome bridges, pollen mother cell division with massive failure, and little fertility. In Doritaenopsis orchids, high pollen viability and high fertility depends on a high frequency of normal tetrads, and low seed set in cross-pollination is predicted with micronuclei in the end products of meiosis. The occurrence of chromosomal aberrations may suggest a process of genome differentiation that could compromise breeding efforts if not taken into consideration. This work was supported by grants from the Council of Agriculture, Taiwan (94AS-1.3.2-FD-Z3 and 95AS-1.3.2-FD-Z3), and by a grant for graduate study to the first author from the International Cooperation and Development Fund of Taiwan, Ministry of Foreign Affairs, Taiwan, Republic of China. UCR::Vicerrectoría de Docencia::Ciencias Agroalimentarias::Facultad de Ciencias Agroalimentarias::Escuela de Agronomía

Anthurium is the largest and most complex genus in the family Araceae. It consists of about 1000 species (Croat 1992). The distribution of this genus extends from northern Mexico and the Greater Antilles to southern Brazil and northern Argentina, and Paraguay (Croat 1983, 1986). The chromosome number of most investigated species is 2n = 30 (Sheffer and Kamemoto 1976; Sheffer and Croat 1983).