Genetic Engineering of Anthurium for Bacterial Disease Resistance

To aid transformation of anthurium, tissue culture and regeneration was achieved through either somatic embryogenesis or shoot regeneration using in vitro grown etiolated internodes, laminae, and root segments. Two gene transfer methods were used to transform anthurium tissues. Using bombardment of DNA-coated microprojectiles into anthurium calli and etiolated internodes, transient expression of S-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) was observed in these tissues. No transformed plants were recovered using this method. Antibacterial genes, including an insect attacin gene (Att), phage P22 gene P13, phage T4 lysozyme gene e, and a gene encoding an analog of insect cecropin B (Shiva-1) were driven by either double CaMV35S or potato wound inducible promoter, in the plant expression vector pBI121. Agrobacterium tumefaciens LBA4404 carrying either pCa2Att, pCa2P13, pCa2T4, pWlAtt or pWlShiva was used for cocultivation with internode or lamina explants of UH965 and UH1060. Following culture on selection media containing kanamycin and carbenicillin or cefotaxime, shoots regenerated from various calli pieces. Kanamycin-resistant plantlets were recovered from UH965 and UH1060 etiolated internode explants cocultivated with Agrobacterium with or without tobacco cell line 'Su' as nurse culture. GUS activity as determined by histochemical staining was absent in the kanamycin-resistant plants evaluated. Western blot analysis of total proteins from lamina calli formed de novo from kanamycin-resistant UH965 plants showed the presence of attacin protein. Polymerase chain reaction was used to amplify DNA fragments from the introduced genes. In six UH965 plants, A tt and nptll genes were amplified in the expected sizes of 546 and 1054 bp, respectively. In UH1060 plants, P13 and nptll genes were amplified. A GUS gene fragment was amplified in one of the UH965 plants. No amplification of the above-mentioned genes was observed in DNA samples of untransformed control plants. Southern hybridizations using amplified sequences from Att, P I3 and NPTH all showed positive hybridization. Kanamycin-resistant UH965 plants with Ca2Att, Ca2P13 or Ca2T4 were challenged with the blight pathogen Xanthomonas campestris pv. dieffenbachiae strain D150 in the petiole end. The result of two challenge experiments indicated that most transgenic UH965 plants were partially resistant and a few were resistant to the blight bacteria

Shoot multiplication of Paphiopedilum orchid through in vitro cutting methods

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

Expression Profiles of Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase Genes in Phalaenopsis, Implications for Regulating the Performance of Crassulacean Acid Metabolism

Phalaenopsis is one of the most important potted plants in the ornamental market of the world. Previous reports implied that crassulacean acid metabolism (CAM) orchids at their young seedling stages might perform C3 or weak CAM photosynthetic pathways, but the detailed molecular evidence is still lacking. In this study, we used a key species in white Phalaenopsis breeding line, Phalaenopsis aphrodite subsp. formosana, to study the ontogenetical changes of CAM performance in Phalaenopsis. Based on the investigations of rhythms of day/night CO2 exchange, malate contents and phosphoenolpyruvate carboxylase (PEPC) activities, it is suggested that a progressive shift from C3 to CAM occurred as the protocorms differentiated the first leaf. To understand the role of phosphoenolpyruvate carboxylase kinase (PEPC kinase) in relation to its target PEPC in CAM performance in Phalaenopsis, the expression profiles of the genes encoding PEPC (PPC) and PEPC kinase (PPCK) were measured in different developmental stages. In Phalaenopsis, two PPC isogenes were constitutively expressed over a 24-h cycle similar to the housekeeping genes in all stages, whereas the significant day/night difference in PaPPCK expression corresponds to the day/night fluctuations in PEPC activity and malate level. These results suggest that the PaPPCK gene product is most likely involved in regulation of CAM performance in different developmental stages of Phalaenopsis seedlings

Trends of Phalaenopsis orchid breeding research

Phalaenopsis is one of the most popular and improtant orchids produecd at commercial scale. New cultivars are produced by selection of seeding plants during their flowering stage. After selection of orchid plants with desirable traits, tissue culture and mass production of young plantlets were achieved through flower stalk node culture in vitro. Cross hybridization by artificial pollination among different parental hybrids usually can lead to capsule set and seed formation. Howerver, breeding barriers are present in Plalaenopsis and its alliance genera with the mechanisms largely remained unknown. Meiotic chromosome behavior of the pollen mother cells (PMC) was investigated to examine whether it is correlated with seed set in different breeding parents of both Doritaenopsis and Phalenopsis orchids. The results showed that high perecntage of normal tetrad, in addition ot regular bivalent chromosome paring, generally suggests high fertility. Unreduced gametes such as dyads were observed during the PMC meiotic process and the number of the dyads could be increased by treating young flower buds with mitotic inhibitors such as colchicines and trifluralin. Through cross Pollination of chemically conditioned pollinia, chance of obtaining polyploidy seedlings is expected to be increased. Through these meiotic analysis and chemical treatment, breeding efficiency of phalanopsis orchids will be increased

Study on Isozyme Variation in Malate Dehydrogenase and 6-phosphogluconate Dehydrogenase of Peach

本試驗利用澱粉膠體電泳探討56個桃品種之MDH及PGD同功異構酶異性，其中MDH共有六種酶型(A, A', B, C, C', D)，低溫需求量高之品種中，有42.4%屬於MDH-B，而低溫需求量少之品種中，則有67%屬於MDH-A。分析MDH遺傳顯示A和C是同一基因座上的兩個共顯性因子。MDH-D型品種之開放授粉後代分離爲A，D及C型，MDH-B型（大久保，白鳳）和MDH-A型(Okinawa, Flordared)雜交可產生A，D，或A，C，或A，B型之後代，有些MDH的條帶可能是受某種修飾因子作用的結果。PGD在土用品種屬於一條型，其他調查過的16個品種均屬於兩條型，進一步分析MDH基因可能有助於解該素與低溫需求性狀的關係。 Isozyme variation in 56 peach varieties was investigated using starch gel electrophoresis. Malate dehydrogenase (MDH) and 6-phosphogluconate dehydrogenase (PGD) were found to be polymorphic. Six banding types (A, A', B, C, C', and D) were observed in MDH. 42.4% of high chilling requirement varieties belonged to MDH-B type, while 67% of low chilling varieties belonged to MDH-A type. Genetic analysis of variable MDH types revealed that A (and probably A') and C (and probably C') were two alleles of a single locus controlling MDH expression. MDH-D was a heterozygote which segregated into types AD, and C. A cross between MDH-B (Okubo or White Phoenix) and MDH-A (Okinawa or Flordared) produced either A, D or A, C, or A, B types, Modification in some MDH isozymes of peach was suggested from this study. PGD showed less variable as compared to MDH. Further characterization of MDH gene (s) may be helpful in elucidating its relationship with chilling requirement in peach

Advances and Perspectives for Polyploidy Breeding in Orchids

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

Cloning of Phytoene Desaturase Gene in Oncidium Gower Ramsey-Orchidaceae and Its Expression Analysis at Different Flower Stages

Oncidium Gower Ramsey is an important ornamental orchid in Taiwan. The bright yellow floral color is due to carotenoid accumulation in the flower tissues. In order to understand the expression of phytoene desaturase (PDS), a key enzyme for the biosynthesis of β-carotene, the cDNA encoding PDS was cloned and characterized during flower development. The full length OgrPDS cDNA contains 2,273 bp, including 1,593 bp open reading frame encoding for a polypeptide of 531 amino acid residues. The 5’ and 3’ untranslated regions (UTR) were 293 and 387 bp, respectively. The deduced amino acid sequence of this PDS has 72-75 % similarity to PDS from other plant species. The relative expression level of the OgrPDS in roots and leaves was investigated. The result showed that the expression level of OgrPDS at different flower developmental stages increased gradually until stage 5 which two- and three- fold higher than leaves and roots, respectively

Isozyme Variation and Inheritance in Inbred Lines of Cucumber (Cucumis sativus L.)

利用澱粉膠體或聚丙烯醘氨膠體電泳分析胡瓜自交系的同功異構酵素變異性，結果有三種酵素具有多型性。蘋果酸去氫酶(MDH)顯示6-8個條帶區，其命名依酵素之移動性朝正極最性者爲1，依此類推。帶區1及3各有兩條移動性不同之條帶(1a, 1b, 3a, 3b)；帶區4在本試驗中顯示一個條帶；帶區2, 5,及6各爲單條帶型；所有自交系均具有不變的帶區2；另外在帶區3、4、5、6也發現有不具酵素活性的空白區，可以是由null allele所控制合成；帶區7及8含一至數個條帶，其解析度較低，無法判斷是否具有變異性；帶區3, 4, 5及6爲子葉特異條帶，在本葉無法偵測到，屬於子葉特異同功異構酶。依各自交系所含不同MDH條帶區之種類，可以分爲一種類型（A，B及C型）。磷酸葡萄糖酸去氫酶(PGD)帶區可分爲PGD-1及PGD-2，快速移動條帶區(1)具有兩個條帶（1a及1b），因此不同自交系可依此將表現型分爲PGD-A或B型，慢速移動條帶區(2)之移動性在所有自交系均相同；在乾燥種子或幼功子葉中均中染出PGD酵素活性。莽草酸去氫酶(SKDH)有兩型，每型均由一群移動較快且較淡色及一條較慢且深染的條帶群組成，兩型之間慢移動條帶的染色較深，且其移動性具有差異，不同自交系亦可分爲SKDH-A或B型，以各型之較慢一條帶可做遺傳分析。將具不同多型性自交系間雜交或回交，以進行遺傳分析，結果顯示Mdh-1，Pgd-1及Skdh-1均各爲由一個基因座的兩個共顯性等位基因所控制；MDH-3, MDH-4條帶區若無null band，則其遺傳模式和上述相同；若具有空白的null band，則其遺傳模式較複雜，目前仍不清礎。具多型性之MDH，PGD及SKDH三種酵素可作爲一代雜交種子純度或育種親本鑑定之標示因子。 Starch gel and polyacrylamide get electrophoresis were used to analyse isozyme polymorphism in 15 inbred lines of cucumber (Cucumis sativys L.) and C. hardwickii inbred. Three enzyme systems were found to be polymorphic, including malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (PGD), and shikimate dehydrogenase (SKDH). A null allele in the MDH-3, MDH-4, and MDH-5 activity zones, winch belong to cotyledon specific isozymes, were detected in some inbred tines. Its mode of inheritance still needs to be elucidated. Genetic analyses of backcross and F2 generations showed that Mdh-1、Pgd-1 and Skdh-1 were all inherited as a single Incas with two alleles, respectively. Mdh-3 locus, when in non-null state, was also controlled by two alleles

Advances and Perspectives for Polyploidy Breeding in Orchids

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 RicaUniversidad de Costa Rica/[814-C0-244]/UCR/Costa RicaUCR::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 Agronomí