Ectopic expression of an orchid (Oncidium Gower Ramsey) AGL6-like gene promotes flowering by activating flowering time genes in Arabidopsis thaliana

An AP1/AGL9 group of MADS box gene, OMADS1, with extensive homology to the Arabidopsis AGAMOUS-like 6 gene (AGL6) was characterized from orchid (Oncidium Gower Ramsey). OMADS1 mRNA was detected in apical meristem and in the lip and carpel of flower. Yeast two-hybrid analysis indicated that OMADS1 is able to strongly interact with OMADS3, a TM6-like protein that was involved in flower formation and floral initiation in orchid. Transgenic Arabidopsis and tobacco ectopically expressed OMADS1 showed similar novel phenotypes by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminacy. In addition, homeotic conversion of sepals into carpel-like structures and petals into staminoid structures were also observed in flowers of 35S::OMADS1 Arabidopsis. This result indicated that OMADS1 was involved in floral formation and initiation in transgenic plants. Further analysis indicated that the expression of flowering time genes FT, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and flower meristem identity genes LEAFY (LFY), APETALA1 (AP1) was significantly up-regulated in 35S::OMADS1 transgenic Arabidopsis plants. Furthermore, ectopic expression of OMADS1 rescued late-flowering phenotype in gi-1, co-3 but not for ft-1 and fwa-1 mutants. These results supported that ectopic expression of OMADS1 influenced flower transition and formation by acting as an activator for FT and SOC1 in Arabidopsis.     

An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation

cDNA for a B group MADS box gene OMADS3 was isolated and characterized from Oncidium Gower Ramsey, an important species of orchid. OMADS3 encoding a 204 amino acid protein showed high sequence homology to both paleoAP3 and TM6 lineage of B group MADS box gene such as monocots AP3 homologue LMADS1 in lily and GDEF1 in Gerbera hybrida. Despite the sequence homology, consensus motifs identified in the C-terminal region of B group genes were absent in OMADS3. Southern analysis indicated that OMADS3 was present in O. Gower Ramsey genome in low copy numbers. Different from most B group genes, OMADS3 mRNA was detected in all four floral organs as well as in vegetative leaves. This is similar to the expression pattern of GDEF1. 35S::OMADS3 transgenic plants showed novel phenotypes by producing terminal flowers similar to those observed in transgenic plants ectopically expressed A functional genes such as AP1. Ectopic expression of OMADS3 cDNA truncated with the MADS box or C terminal region in Arabidopsis generated novel ap2-like flowers in which sepals and petals were converted into carpel-like and stamen-like structures. Yeast two-hybrid analysis indicated that OMADS3 is able to strongly form homodimers. Our results suggested that OMADS3 might represent an ancestral form of TM6-like gene which was conserved in monocots with a function similar to A functional gene in regulating flower formation as well as floral initiation.     

The Sweet Pepper Ferredoxin-Like Protein (pflp) Conferred Resistance Against Soft Rot Disease in Oncidium Orchid

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.     

Ectopic expression of two MADS box genes from orchid (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey) and lily (<Emphasis Type="Italic">Lilium longiflorum)</Emphasis> alters flower transition and formation in <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis>

Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks’ duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation.     

Agrobacterium tumefaciens-mediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1-1

Oncidium and Odontoglosum orchid species have reduced display lives and are thus commercially less important than Phalaenopsis. One approach to prolonging display life permanently is to transform Oncidium and Odontoglossum with the ethylene receptor mutant gene etr1-1 from Arabidopsis under control of a flower specific promoter; this should reduce their sensitivity to exogenous ethylene. To achieve this it will be necessary to establish an efficient regeneration protocol using somatic embryogenesis and a routine Agrobacterium tumefaciens-mediated transformation procedure. Protocorm-like bodies (PLBs) of both orchid genera were regenerated from leaf tip explants. Leaf tips and PLBs, cultured in liquid and solid media, were compared as targets for genetic transformation. No transgenic shoots were obtained from leaf tips, while PLBs of Oncidium and Odontoglossum cultured on solid medium were successfully transformed with an expression vector containing nptII and gus genes driven by the cauliflower mosaic virus (CaMV) 35S promoter. Applying the A. tumefaciens strain EHA 105, transformation efficiencies of 1.3–2.7% were achieved for the investigated genotypes. Transformation with etr1-1 gene was achieved subsequently. Oncidium ‘Sweet Sugar’ has been successfully transformed and validated by PCR and Southern analysis.     

Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey)

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for orchid Oncidium Gower Ramsey. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose selective medium. Genetically transformed plants of Oncidium were regenerated after cocultivating protocorm-like bodies with Agrobacterium tumefaciens strain GV3101 containing the vectors pEPYON-42P and pEPYON-42H with 35S::PMI and 35S::HPTII genes respectively. We observed that 35S::PMI (pEPYON-42P) produced high rate (27 plants) of mannose resistant transgenic plants compared to 35S::HPTII (pEPYON-42H) in which only fourteen hygromycin resistant transgenic plants were obtained. Mannose resistant transgenic plants were confirmed by PCR and Southern blot. The pmi gene expression in 35S::PMI (pEPYON-42P) transgenic plants was confirmed by RT-PCR. Furthermore, the duration of regeneration time of transgenic plants was significantly shorter in mannose selected system (4 months) than in hygromycin selected system (8 months). The pmi/mannose selection system is shown to be highly efficient for producing transgenic O. Gower Ramsey without using antibiotics or herbicides. For the first time, the pmi/mannose-based “positive” selection system has been used to obtain genetically engineered O. Gower Ramsey.     

Developmental and LED Light Source Modulation of Carotenogenic Gene Expression in Oncidium Gower Ramsey Flowers

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.     

Modification of flower colour by suppressing β‐ring carotene hydroxylase genes in Oncidium

Oncidium ‘Gower Ramsey’ (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The β‐ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH‐A2, BCH‐B2 and BCH‐C2 isolated from Onc. GR, to analyse their roles in flower colour. RT‐PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH‐B2 remained constant while that of BCH‐A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down‐regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white‐yellow. BCH‐A2 and BCH‐B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down‐regulation of BCH‐A2 and BCH‐B2 changed the flower colour from bright yellow to light and white‐yellow.     

Genetic transformation of Cymbidium orchid by particle bombardment

 A protocol is presented for genetically engineering Cymbidium orchid using particle bombardment. This protocol enabled the routine transformation of orchid plants that were previously difficult to transform. Liquid culture was used to generate a large number of protocorm-like bodies (PLBs) to be bombarded and to promote continued development of the bombarded meristematic tissue. Plasmid DNA (pKH200) carrying the GUS-INT and NPTII genes flanked by tobacco matrix attachment regions was introduced into the meristematic cells of PLBs by particle acceleration. The transformed PLBs were proliferated and selected for kanamycin resistance conferred by the introduced NPTII gene. Shoot regeneration was then induced from the kanamycin-resistant PLBs, and transgenic plantlets were produced. Both the kanamycin-resistant PLBs and regenerated shoots expressed the GUS-INT gene. The presence of the introduced gene in the transformed orchid plants was confirmed by PCR analysis, sequencing and Southern blot analysis of the PCR product. The recovered transgenic plants were established in soil and acclimatized in the greenhouse. Received: 20 July 1998 / Revision received: 2 December 1998 / Accepted: 17 December 1998     

Molecular characterization of the PpMADS1 gene from peach

PpMADS1, a member of the euAP1 clade of the class A genes, was previously cloned from peach. In this study, PpMADS1 was constitutively expressed in Arabidopsis thaliana to study its function in plant development. The transgenic A. thaliana plants containing 35S::PpMADS1 showed severe phenotype variation including early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, production of higher number of carpels, petals, and stamens than non-transgenic plants, and prevention of pod shatter. Significantly, the transgenic plants produced more than one silique from a single flower. The results obtained by using cDNA microarray and real-time PCR analyses in the transgenic Arabidopsis indicated that PpMADS1 might play dual roles in regulating the floral meristem development by activating or repressing different sets of genes that would determine the different fate of a floral meristem. In addition, the PpMADS1 gene promoter was further cloned, and deletion analyses were conducted by using fused GUS as a reporter gene in transgenic A. thaliana. Histochemical staining of different organs from transgenic plants revealed the region between ?197 and ?454?bp was specific for GUS expression in flower primordium, and the region between ?454 and ?678?bp was specific for GUS expression in sepals and petals. In contrast, a negative regulatory element present between ?678 and ?978?bp could suppress GUS expression in filament.     

Delay of flower senescence and abscission in Arabidopsis transformed with an FOREVER YOUNG FLOWER homolog from Oncidium orchid

The ectopic expression of FOREVER YOUNG FLOWER (FYF), a MADS box gene in Arabidopsis, caused significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. It was proposed that the function of the FYF gene was related to the regulation of senescence and abscission. This hypothesis was further supported by one line of evidence reported in this study. The evidence is the similar delay of flower senescence and abscission observed in transgenic Arabidopsis ectopically expressing OnFYF, an FYF homolog from the Oncidium orchid, a monocot. This data suggested that the function of FYF homologs in regulating flower senescence and abscission was highly conserved in both dicot and monocot plants.     

Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in <Emphasis Type="Italic">Oncidium</Emphasis>

Traditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby OM8 (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3- to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and -glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation.     

Chemical study of the flowers of the orchid Oncidium baueri Lindley and their visiting bees Trigona spinipes Fabricius

Oncidium baueri Lindley is a South American orchid characterized by the production of numerous yellow-brown flowers, arranged in inflorescences that reach up to four meters long. Herein, the chemical study of the flowers led to the identification of compounds present in the floral oil, such as long chain acids and methyl esters, including the first report of the occurrence of rare compound byrsonic acid in orchids in the free form. In addition to this, steroids and the flavonoid glycosides acacetin-7-O-rutinoside, pectolinarin, oncibauerins A and B were identified in large amounts. Finally, the flower-insect visit by Trigona spinipes Fabricius bees was also studied. Video analyses of the process revealed that the bees spend a long time collecting material from the flower labellum callus. The chemical analyses of bees and flowers showed the presence of palmitic acid and stearic acid in both samples, leading to suspicious about the attraction of bees by flowers oils as floral rewards.     

Molecular Cloning of ADP-Glucose Pyrophosphorylase Large Subunit cDNA from Oncidium

A full-length cDNA for ADP-glucose pyrophosphorylase large subunit (AGPL) was isolated from tropical epiphytic orchid Oncidium hybrid Goldiana. The cDNA was 1754 bp in length with an open reading frame of 1551 bp encoding 517 amino acids. The deduced amino acid sequence showed 73 % identity with those of potato isoform 3 (AGPL3) and Arabidopsis thaliana isoform 1 (AGPL1), 71 % identity with that of barley isoform BLPL. RT-PCR analysis showed that AGPL was expressed in mature leaf, immature leaf, developing inflorescence and flower of Oncidium. No expression was detected in roots.     

Gene stacking in <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchid enhances dual tolerance to pathogen attack

Cymbidium Mosaic Virus (CymMV) and Erwinia carotovora have been reported to cause severe damage to orchid plants. To enhance the resistance of orchids to both viral and bacterial phytopathogens, gene stacking was applied on Phalaenopsis orchid by double transformation. PLBs originally transformed with CymMV coat protein cDNA (CP) were then re-transformed with sweet pepper ferredoxin-like protein cDNA (Pflp) by Agrobacterium tumefaciens, to enable expression of dual (viral and bacterial) disease resistant traits. A non-antibiotic selection procedure in the second transformation minimized the potential rate of ‘stacking’ antibiotic genes in the orchid gene pool. Transgene integration in transgenic Phalaenopsis lines was confirmed by Southern blot analysis for both CP and pflp genes. Expression of transgenes was detected by northern blot analysis, and disease resistant assays revealed that transgenic lines exhibited enhanced resistance to CymMV and E. carotovora. This is the first report describing a transgenic Phalaenopsis orchid with dual resistance to phytopathogens.     

Early flowering and increased expression of a FLOWERING LOCUS T-like gene in chrysanthemum transformed with a mutated ethylene receptor gene mDG-ERS1(etr1-4)

Ethylene has an inhibitory effect on flowering in a short-day (SD) plant chrysanthemum (Chrysanthemum morifolium Ramat.). In this study, we used a hexaploid chrysanthemum ??Sei-Marine?? and found that its transgenic lines transformed with a mutated ethylene receptor gene mDG-ERS1(etr1?C4), which conferred reduced ethylene sensitivity (J. Plant Biol. 51: 424?C427, 2008), opened flowers earlier than the non-transformed control. We examined whether the accelerated flower induction in the transformant occurred through the enhanced expression of chrysanthemum genes homologous to FLOWERING LOCUS T (FT), a floral inducer gene in Arabidopsis. We cloned three cDNAs for FT homologs (CmFTL1, CmFTL2, and CmFTL3) from ??Sei-Marine??. CmFTL2 putatively encodes a non-functional gene product due to a frame shift caused by a 2 bp-deletion in the coding region. RT-PCR analysis revealed differential expression patterns of CmFTL genes in the transgenic and control lines, suggesting that these genes might be under the control of ethylene. CmFTL1/2 mRNA level was lower in a SD condition than a long-day (LD) condition. CmFTL3 mRNA accumulated abundantly under SD condition as compared with LD condition in the transgenic line. These results suggest the association of increased expression of CmFTL3 gene with the accelerated flowering in the transgenic line with reduced ethylene sensitivity.     

Induction of petal-bearing embryos from root-derived callus of Oncidium ‘Gower Ramsey’

A repeatable in vitro culture method was established and it could induce a series of abnormal embryos by which their scale leaves were substituted by petals. These petal-bearing embryos were derived from long-term root calli of an orchid cultivar, Oncidium ??Gower Ramsey??. The calli were induced and subcultured on a modified 1/2MS medium supplemented with five combinations of TDZ and dicamba. When 1-year-old callus, which induced and subcultured at 3?mg/l TDZ and 5?mg/l dicamba (line 13 callus), was transferred onto 1/2MS medium supplemented with 0.1?ml/l NAA and 3?mg/l TDZ, it gave the highest number of petal-bearing embryos. However, line 13 root explants gave one of the lowest percentage of callus formation (12.5%) and the number of somatic embryos per callus was also one of the lowest (along with lines 10, 11 and 12). The efficiency of embryogenesis decreased with age of callus and the significant decrease was started from the third year of culturing. Flowering characteristics of plantlets from normal embryos and petal-bearing embryos were both evaluated after 3?years of culture in the greenhouse. Parameters including length of the longest inflorescence, numbers of flowers per plant, length of flowers and width of flowers were all not significantly different between abnormal embryo-derived plants and normal embryo-derived plants.     

Direct somatic embryogenesis on leaf explants of Oncidium Gower Ramsey and subsequent plant regeneration

 Segments taken from young leaves of an orchid (Oncidium Gower Ramsey) produced clusters of somatic embryos directly from epidermal and mesophyll cells of leaf tips and wound surfaces without an intervening callus within 1 month when cultured on a Gelrite^TM-gelled 1/2-MS basal medium supplemented with a low dosage (0.3–1 mg/l) of thidiazuron. Subculturing of these embryo clusters produced more embryos and subsequent plantlet formation on the same medium. The high-frequency embryogenesis of these leaf cells in this orchid is strong evidence of their totipotency, and further modification of the protocol for plant formation could be useful for the mass propagation and transformation of selected elite lines. Received: 16 September 1998 / Revision received: 16 February 1999 / Accepted: 26 February 1999