4. Orchid breeding_Recent advances in biotechnology and
Transcription
4. Orchid breeding_Recent advances in biotechnology and
3 Feb. 2015 Orchid breeding: Recent advances in biotechnology and considerations So-Young Park, Ph.D Department of Horticultural Science, Chungbuk National University Republic of Korea 1 Contents • Introduction • Micropropagation : Regeneration and Development • Breeding by in vitro biotechnology • Consideration : Somaclonal variation • Conclusions 2 3 INTRODUCTION Orchid Industry in Korea Total area utilized for floriculture was around 6,640 ha and the economic value reached US$ 720 million. Among them 29% of the total pot flower cultivation area is allocated for orchid production. The value of orchid production reached up 36.8% of the total pot flower production value in Korea. The number of orchid growers is around 804 (7.5%) out of 10,690 flower growers. Orchid production is one of the most representative bio-agricultural industries in Korea. 4 INTRODUCTION Orchid Industry in Korea (1,000$) Import Export (1,000$) 5 INTRODUCTION Orchid Industry in Korea Orchid production (RDA, 2009) 6 INTRODUCTION Orchid Cultivation in Korea Kyeongki Gangwon-do 38.8 % (97.9 ha) Incheon Chungcheongbuk-do 3.6%(9.0ha) Gyeongsangbuk-do Daejeon Chungnam 20.3 % (51.4 ha) Jeollabuk-do Daegu Ulsan Kyeongnam 8.6 % (21.6 ha) Gwangju Jeonnam Jeonnam 6.7 % (16.8 ha) Jeju-do Jeju 11.9 % (30.1 ha) 7 Micropropagation - Regeneration and Development - 8 Conventional propagation by cutting and division Mother plants for flowers and pseudo bulbs for propagation Dendrobium-Phalaenopsis propagation (TOC group in Thailand) 9 Dendrobium-Phalaenopsis propagation (TOC group in Thailand) Conventional propagation by cutting and division Dendrobium-Phalaenopsis propagation (TOC group in Thailand) New shoot production from old pseudobulb 10 Dendrobium-Phalaenopsis propagation (TOC group in Thailand) 11 Dendrobium-Phalaenopsis propagation (TOC group in Thailand) 12 Dendrobium-Phalaenopsis propagation (TOC group in Thailand) 13 Micropropagation by seed germination Orchid capsule and seeds Protocorm In vitro seed germination 14 Dendrobium-Phalaenopsis propagation (TOC group in Thailand) 15 Micropropagation by clonal propagation * Protocormlike body PLB propagation (Paek et al. 2011, Plant Embryo Culture) Fig. Vegetative propagation of elite orchid via PLB multiplication. 16 Concept of mass production system via micropropagation Breeding by in vitro biotechnology 1. 2. 3. 4. Micropropagation Polyploidy breeding Mutation breeding Genetic transformation 18 Micropropagation • Mass propagation of elite genotype by selection from seedling populations - Flower color, disease resistance, flowering time.. • Regeneration ability of plant • Essential of plant cell and tissue culture technique Rapid establishment of new variety 19 Orchid MICROPROPAGATION (Phalaenopsis) Proliferation via PLB/multiple-shoots PPF (µmol·m-2·s-1) 50 100 150 200 300 M M 1H 1M 3H 3M 5H 5M 7H C1 1H 1M 3H 3M 5H C4 Acclimatization and greenhouse cultivation 7M 5M 7H 7M 1H 1M 3H 3M 5H C2 1H 1M 3H 3M 5H 5M 7H 7M 5M 7H 7M C3 Variant detection 20 Mass production via solid/liquid culture Ploidy breeding • Polyploidy is plants or cells containing more than two paired sets of chromosomes. • Polyploidy is responsible for increases in cell size, a characteristic that leads to larger vegetative and reproductive organs (Kim et al, 2004). 2n 4n Phalaenopsis equestris var cyanochilus 21 Solanum commersonii (Riccardo et al, 2012) Characteristics of ploidy orchid Polyploidy orchid Chemicals for chromosome doubling 1. Colchicine(C22H25NO6) • Narcotic alkaloid extraction from Colchicum autumnale • Inhibition of microtubules during mitosis of plant cell 2. Oryzalin(C12H18N4O6S) • A kind of herbiside • The disruption of microtubules during mitosis 22 Cymbidium Show Girl ‘Silky’ Characteristics of ploidy orchid Breeding in National Institute of Horticultural & Herbal Science Species : Cymbidium Elma ‘Orient Toyo’, C. Golden Elf ‘Sundust’, C. Show girl ‘silky’ Diploid and tetraploid plants by treatments of Colchicine 100-500 mg/L for 1-2 week Cymbidium Elma ‘Orient Toyo’ Cymbidium Golden Elf ‘Sundust’ 23 Characteristics of ploidy orchid Analysis of DNA content for ploidy determination Treatment of colchicine and oryzalin Flow cytometry Extraction Chopping and extraction of nuclei Filtering Nuclei staining 24 Characteristics of ploidy orchid Cymbidium Elma ‘Orient Toyo’ Number of nuclei 80 2n Diploid Tetraploid 60 4n 40 4n 20 8n 50 100 150 200 50 100 150 200 Relative DNA content Fig. Flow cytometric DNA histograms of diploid and tetraploid of Cymbidium Elma ‘Orient Toyo’. 25 Characteristics of ploidy orchid Cymbidium Golden Elf ‘ Sundust’ Number of nuclei 80 Diploid 2n Tetraploid 60 4n 40 4n 20 8n 50 100 150 200 50 100 150 200 Relative DNA content Fig. Flow cytometric DNA histograms of diploid and tetraploid of Cymbidium 26 Golden Elf ‘Sundust’. Characteristics of ploidy orchid A B C Fig. Control plants (A), chromosome doubled plants (B), ploidy chimera plants (C) of Cymbidium Show Girl ‘Silky’. 27 Characteristics of ploidy orchid Cymbidium Show Girl ‘Silky’ A(3x) B(6x) Fig. Guard cells of control and chromosome doubled plant of Cymbidium Show Girl ‘Silky’. 400× magnification. Bars = 40㎛. 28 Characteristics of ploidy orchid Flower morphology by ploidy level A Diploid B Tetraploid Fig. Diploid C and tetraploid D flowers of Cymbidium Elma ‘Orient Toyo’. (Hwang et al. unpublished data) 29 Characteristics of ploidy orchid Flower morphology by ploidy level Lateral sepal (upper) Petal Stigma Lip Lateral sepal (under) Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Elma ‘Orient Toyo’. 30 (Hwang et al. unpublished data) Characteristics of ploidy orchid Flower morphology by ploidy level Diploid Tetraploid Fig. Diploid A and tetraploid B flowers Cymbidium Golden Elf ‘Sundust’. (Hwang et al. unpublished data) 31 Characteristics of ploidy orchid Flower morphology by ploidy level Lateral sepal (upper) Petal Stigma Lip Lateral sepal (under) Fig. Diploid (Left) and tetraploid (Right) floral parts of Cymbidium Golden Elf ‘Sundust’. (Hwang et al. unpublished data) 32 Characteristics of ploidy orchid Fragrance pattern in ploidy plant • Analysis of fragrance pattern using electronic nose - MOS : High similarity with human olfactory sense - Use : Fox2000 with Sensor Array System (Alpha M.O.S) • Materials : Cymbidium Golden Elf ‘Sundust’ di-, tetraploid • Alpha soft model : PCA (Principle component analysis), DFA (Discriminant factor analysis) 33 Characteristics of ploidy orchid Scent pattern of di- and tetraploid flower (PCA) ■ Control (air) ▲ Sundust 2n=2x ● Sundust 2n=4x Fig. Principle component analysis (PCA) plot of the scent from the 2n and 4n flowers in C. Golden Elf ‘Sundust’ using the electronic nose. (Hwang et al. unpublished data) 34 Characteristics of ploidy orchid Scent pattern of di- and tetraploid flower (DFA) ■ Control (air) ▲ Sundust 2n=2x ● Sundust 2n=4x Fig. Discriminant factor analysis (DFA) plot of the scent from the 2n and 4n flowers in C. Golden Elf ‘Sundust’ using the electronic nose. (Hwang et al. unpublished data) 35 Mutation breeding • X-ray, gamma ray irradiation, and chemical treatments • Powerful tools for plant breeding as well as for physiological and molecular studies • Gamma (y) radiation - a type of ionizing radiation - produce free radicals in cells which damage or modify DNA of plant cells and affect differently the morphology 36 Mutation breeding 50 Gy WT Gamma ray effect on damaging DNA (Wi et al., 2007) 37 Mother plants Mutants 38 Mutation breeding (Taheri et al. 2014) Curcuma alismatifolia varieties by gamma ray 39 Mutation breeding of orchid Genetic mutation Some genetic variants are popular and useful Varients of Aerides japonicum obtained from asymbiotic seed cultures Very popular in special grower with high price 40 Genetic transformation 41 Genetic transformation of orchid Genetic transformation Delivering the gene to the plant • Transformation cassettes are developed in the lab • They are then introduced into a plant • Two major delivery methods - Agrobacterium - Gene Gun (Particle bombardment) Agrobacterium Gene gun 42 Genetic transformation of orchid Genetic transformation by Agrobacterium 43 World Orchid Exhibition 2012. Okinawa in Japan Genetic transformation of orchid 44 World Orchid Exhibition 2012. Okinawa in Japan Genetic transformation of orchid 45 Genetic transformation of orchid 46 Control of flowering time by transformation FT2 Shortens the Juvenile Phase and Promotes Seasonal Flowering HindIII HindIII XbaI Sac I RB Pnos NPT II Tnos (Hsu et al., 2008, Plant Cell) CaMV 35S PagFT2 EcoRI LB Tnos (Igasaki et al., 2008, Plant Cell Physiol) 47 Control of flowering time by transformation FT2 Shortens the Juvenile Phase and Promotes Seasonal Flowering HindIII HindIII XbaI Pnos NPT II Tnos CaMV 35S WT - Phalaenopsis cv ‘Spring dancer’ - Destination vector pBI121 - 35S::FT2 EcoRI Sac I RB PagFT2 LB Tnos Putative TP Selection of transgenics on 50 μM Kanamycin (Park et al, unpublished data) 48 Somaclonal Variation - Things to be consider - 49 SOMACLONAL VARIATION 50 Somaclonal variation of orchid Somaclonal variation What’s somaclonal variation? The variation seen in plants that have been produced by long-term micropropagation Somaclonal variation - Genetic variation : caused by DNA changes - Epigenetic variation : caused by temporary phenotypic changes 51 Somaclonal variation of orchid Problems of somaclonal variation Morphological changes during floral development in in vitro clonally propagated Orchid Decrease of plant quality in uniformity Normal plant Variations in flower morphology 52 Somaclonal variation of orchid What cause somaclonal variation? Micropropagation Somaclonal variants - Changes in biochemical and molecular events - Changes in DNA methylation pattern - Activation of transposable elements or retroelements Mother plant - Chromosome remodeling Clonally propagated plant 53 Somaclonal variation of orchid Somaclonal variation – polyploid Number of nuclei Number of nuclei 2C Diploid 4C 8C A Tetraploid 4C 8C 16C B DNA relative fluorescence intensity Easily detected during in vitro culture process 54 Somaclonal variation of orchid 55 Somaclonal variation of orchid Various mutants in flowers (Phalaenopsis) P. Baldans Kalaidescop 56 Somaclonal variation of orchid Detection of epigenetic variation in tissue-culture-derived plants of Doritaenopsis RAPD result Methylation pattern analysis (Park et al. 2009) 57 Morphological changes of flowers in P. Wedding Promenade WP-N WP-V1 WP-V2 WP-V3 58 Morphological changes of flowers in P. Spring Dancer SD-N SD-V1 SD-V2 59 Morphological characteristics of leaf and flower stalk B A D C SD-N SD-N SD-V1 SD-V2 SD-V1 SD-V2 Fig. Leaves and flower stalks of P. Spring Dancer (SD) : (A) Leaves, (B) Surface of leaves, (C) Flower stalks, (D) Thickness of flower stalks. (Bar of A,B,D = 1cm, Bar of C = 5cm) 60 Somaclonal variation of orchid Morphological characteristics of flowers A B 3.0 SD-N SD-V1 SD-V2 Parameter (cm) 2.5 2.0 a ab a a SD-N a b 1.5 1.0 SD-V1 0.5 0.0 Width of petals Length of petals SD-V2 Fig. Width and length of petals(A) and flowers (B) in normal and variants in P. Spring Dancer (SD). (Unpublished data) 61 Somaclonal variation of orchid Morphological characteristics of leaves A C Parameter (cm) 18 15 SD-N SD-V1 a 12 b 9 6 a ab 3 a b 0 Width of leaves Length of leaves 50 Leaf area (cm2) B 40 a a b 30 SD-N 20 10 0 SD-N SD-V1 SD-V2 SD-V1 SD-V2 Fig. Width and length of leaves (A) and leaf area(B) and leaves (C) in normal and variants in P. Spring Dancer (SD). (Unpublished data) 62 Somaclonal variation of orchid Morphological characteristics of flowers and leaves A Petal 12 SD-N SD-V1 SD-V2 Thickness (mm) 10 a 8 Leaf 6 b 4 c a b 2 c b b a 0 Petals Flower stalks Flower stalk Leaves SD-N SD-V1 SD-V2 Fig. Thickness of petal, flower stalk and leaf in P. Spring Dancer (SD). (Bar of petal and leaf = 0.5 mm, bar of flower stalk = 1 mm). (Unpublished data) 63 Somaclonal variation of orchid Ploidy and endoreduplication analysis A N V1 4C 8C 2C 16 C 4C V2 8C 8C 4C 16 C B N 2C V1 2C 4C 4C V2 2C 8C 4C Fig. The nuclear DNA content and distribution of endopolyploid nuclei in petals (A) and leaves (B) of P. Spring Dancer (SD). (Unpublished data) 64 Somaclonal variation of orchid What determine floral structure? Genetic mechanisms that establish floral organ identity MADS-box genes involved most central genes in floral organ identity 65 Somaclonal variation of orchid PMADS4 expression in flowers and leaves Normalized expression 6 5 Leaf Flower 4 Actin 3 PMADS4 2 1 0 N V1 V2 N V1 V2 Fig. Real-Time PCR analysis for PMADS4 expression level of normal and variant’s flowers and leaves in P. Spring Dancer (SD). (Unpublished data) 66 Somaclonal variation of orchid PMADS4 expression in flowers Normalized expression 16 14 12 10 8 6 4 2 0 P S S P S L Actin Sepal Petal N V1 Sepal V2 N V1 Petal V2 N V1 V2 Lip Lip Fig. Real-Time PCR and electrophoresis analysis for PMADS4 expression level of normal and variant’s flower organs in P. Spring Dancer (SD). (Unpublished data) 67 Conclusions Things to be improved in orchid breeding & production Stable production of high-quality clones by micromropagation Development of new varieties for shorten juvenile phase Year-round culture system by applying hydroponic cultures Breeding new varieties by application of biotechnology Strict control of disease and insects 68 International Orchid Conference World Orchid Conference (WOC) 2011 20th WOC in Singapore 69 International Orchid Conference Asia Pacific Orchid Conference (APOC) 2016 APOC in Bangkok, Thailand 70 2012 APOC in Okinawa, Japan International Orchid Conference International Orchid Symposium (IOS) 2018 3rd IOS in Korea 2014 2nd IOS in Bangkok, Thailand 71 Thank you! 72
Similar documents
Efficient adventitious shoot regeneration from shoot tip culture of
Relative DNA content