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Modern approaches for improving qualitative and quantitative traits in ornamental flowers
- 1. Credit seminar By: Tanya Thakur L-2012-A-27-D Modern approaches for improvement of qualitative and quantitative traits in ornamental flowers
- 2. Qualitative and Quantitative Traits Character Qualitative Quantitative Genetic control Two or many alleles of a single gene or few major genes Many alleles of many gene Inheritance Monogenic Polygenic Environmental effects Little or no modifications More influenced Classified as Grouped into categories (discontinuous variability) Continuous range of variability from one to second extreme Examples Black or white colour, disease susceptibility or resistance, fragrant or non fragrant, flower form Plant height, flower and seed yield Poehlman and Borthakur, 1959
- 3. Crop Qualitative Quantitative Reference Rose Glossy leaf, climbing habit, double flower, mildew resistance Vigour, fragrance, thorniness, leaf width, shape of bud and open flower Wylie, 1954 Lily Lily mottle virus resistance in Asiatic lily Van Heusden et al, 2002 Carnation Doubleness Imai, 1938 Lutescent seedling, club neck, dwarfness Bhatt, 1989 Gladiolus Colour of florets Flower earliness, plant height, spike length , no of florets per spike Cohat, 1988 Marigold Double flower Flatness of florets Flower colour Flower and seed yield, plant height Towner, 1961 Punnet, 1924 Singh and Swarup, 1973 Qualitative and Quantitative Traits in Ornamental Crops
- 4. Approaches for Improvement of Traits Breeding methods Conventional Non Conventional Hybridization Mutation Biotechnological approaches Genetic engineering Tissue culture X
- 5. • Fusarium resistance in gladiolus Beauty Spot x Pssitacinus hybrid Hybrid-82-10-90 Watermelon Pink x Lady Jhon Hybrid 82-7-59 Watermelon Pink x Mansoer Hybrid 82-18-16 (Negi et al, 1991) • Bacterial wilt resistance in carnation Super Gold x Dianthus capitatus (spray) 91 BO4-2 (Onozaki et al, 1998) Tolerant Resistance
- 6. • Fusarium resistance in lily L. dauricum x L. longiflorum cv. Gelria (Resistant) (Susceptible) Hybrid (Resistant) (Loffler et al, 1996) • Fragrance in rose Seedling x Prima Ballerina Fragrant Cloud (super fragrance) (Wheatcroft, 1970) Both parents should be essentially fragrant for fragrance in hybrid (Swarup et al, 1973)
- 7. • A new pink mini cut flower gerbera cv. 'Summer Ring' Grandeur x Nova Zembla Summer Ring (Chung et al, 2008) • First summer flowering gladiolus Gladiolus natalensis x G. oppositiflorus (2n=90) (2n=30) Gladiolus gandavensis (2n=60) (De, 2011)
- 8. • Marigold hybrids released by IARI Cracker Jack x Golden Jubilee Golden Yellow x Sun Giant Pusa Narangi Gainda Pusa Basanti Gainda (loose flower and carotenoid) (pots and beds) AICRP on Floriculture, 2007
- 9. Dianthus plumarius Yellow carnation Chalcones •Inter specific hybridization in Dianthus plumarius – Garden pink for transferring yellow colour Gatt et al, 1998 X X Dianthus plumarius D. knappii Flavone and flavonol glucosides
- 10. Hybrids released by PAU, Ludhiana Hybrid Parentage Flower colour Diseases tolerance Punjab Glad- 1 Happy End x True Yellow Orange with yellow center Moderately against Botrytis and Fusarium Punjab Flame Sylvia x White Prosperity Carmine pink Moderately against Botrytis Punjab Glance Happy End x Yellow Stone Orange with yellow centre Moderately against Botrytis Punjab Lemon Delight Jacksonville Gold x White Prosperity Lemon yellow Moderately against Botrytis Punjab Pink Elegance Suchitra x White Prosperity Soft pink Moderately against Botrytis AICRP on Floriculture, 2014 Gladiolus
- 11. Punjab Glance Punjab Lemon Delight
- 12. Punjab Pink Elegance Punjab Flame Punjab Glad 1
- 13. Chrysanthemum Hybrid Parentage Flower colour Flower type Flower size (cm) Winter Queen White Bouquet x Flirt Pink Spoon 9.1 Yellow Delight White Bouquet x Gul-e-Sahir Yellow Pompon 5.2 Royal Purple Bindiya x Rage Purple-pink Anemone 5.25 Autumn Joy White Bouquet x Flirt Shiny pink Decorative 6.6 Anmol Puncho x Rage Yellow Anemone 4.0 AICRP on Floriculture, 2014
- 14. Winter Queen Anmol Yellow Delight Royal Purple Autumn Joy
- 16. Crop Cultivar Mutagen Parent Earlier colour Changed colour Chrysanthemum 1. Agnirekha Gamma rays D-5 Magnolia purple Erythrite red 2. Alankar Gamma rays D-5 Magnolia purple Spanish orange 3. Batik Gamma rays Flirt Red Yellow stripes on red background 4. Tulika Gamma rays M-24 Purple 5. Surekha Yellow Gamma rays Surekha Ruby red Yellow 6. Raktima Gamma rays Shyamal Purple Crimson Bougainvillea 1. Mahara variegata Gamma rays Mahara Green leaves Variegated leaves 2. Jaya Gamma rays Jayalakshmi - Purple bracts 3. Suvarna Gamma rays Ceylon Single Altered flower colour Varieties evolved through Mutation
- 17. Crop Cultivar Mutagen Parent Earlier colour Changed colour Rose 1.Abhisarika Gamma rays Kiss of fire Normal Striped 2. Curio Gamma rays Imperator - Cherry red 3. Light Pink Prize Gamma rays First Prize Light red and deep pink Light Pink 4.Sharada Gamma rays Queen Elizabeth Carmine rose Light pink 5. Madhosh H.T EMS Gulzar - Mauve coloured stripes against deep red base Gladiolus 1. Shobha Gamma rays Wild Rose Roseine purple Shell pink 2. Tambari Gamma rays Oscar Single Altered flower colour Source: http://mvgs.iaea.org Chrysanthemum variety from IARI through Mutation induced by Gamma rays 1. Pusa Anmol (Ajay) 2. Pusa Centenary (Thai Chen Queen) 3. Pusa Kesari (Thai Chen Queen) 4. Pusa Arunodaya (Thai Chen Queen)
- 18. ROLE OF BIOTECHNOLOGY IN IMPROVEMENT
- 19. Recombinant DNA Clones Gene cloning (PCR) TRANSGENIC PLANT Biotechnology Genetic engineering Tissue culture
- 20. ROLE OF GENETIC ENGINEERING IN IMPROVEMENT OF TRAITS IN ORNAMENTALS Biotic and abiotic stress resistance Development of new novel flower colors Flowers: improved shape, size and form improved floral fragrance increased vase life
- 21. Resistance to Biotic Stress Gene Resistance against Bt gene Insects- Lepidoptera, coleoptera & diptera ipt gene (isopentenyl transferase) Insect-Tobacco hornworm & peach aphid CpTI (cowpea trypsin inhibitor) Insect- Lepidoptera, coleoptera, orthopteran Serine proteinase inhibitor Insect- Whitefly Chitinase Fungus- Rhizoctonia, Fusarium Osmotin Fungus- Phytophthora 1,3 -β glucanase and stilbene synthase Fungus- Botrytis cinerea Cercopin Bacteria- Pseudomonas Coat protein gene Virus- TMV
- 22. • Insect resistance Modified delta-endotoxin gene (modified cry1Ab of Bt) Chrysanthemum cv. 'Shuho-no-chikara Resistance against lepidopteran insects (Shinoyama et al, 2003) • Virus resistance Schizosaccharomyces pombe RNA-specific ribonuclease gene (pacl) Chrysanthemum Chrysanthemum stunt viroid (CSVd) resistance (Ogawa et al, 2004)
- 23. Fungal and Bacterial Resistance Host Gene transferred Resistance Reference Carnation Osmotin, PR-1 and/or chitinase Fusarium oxysporum f. sp. Dianthi Zuker et al, 2001 Chrysanthemum Rice chitinase Botrytis Takatsu et al, 1999 Rose Anti-microbial peptide Ace-AMP I Sphaerotheca pannosa Li et al, 2003 Geranium Onion anti-microbial protein Botrytis cinerea and bacteria Bi et al 1999 Geranium Cercopin Xanthomonas Renou et al, 2000 Oncidium orchids Sweet pepper ferredoxin-like protein Bacterial soft rot caused by Erwinia carotovora Liau et al, 2003 Rose Rice chitinase Diplocarpon rosae Marchant et al, 1998
- 24. Resistance to Abiotic Stress - Heat stress Expression of At DREB1A gene from Arabidopsis in Chrysanthemum Transgenic plant and Wild Type (WT) plant exposed to 45°C as heat stress for 36 hours 3 week after heat stress – 70 % transgenic plant survived 20 % Wild type plant survived Leaf electrolyte leaking significantly lower in transgenic plants than Wild type Hong et al, 2009
- 25. Flower colour pigments Flavonoid Betalain Carotenoid Anthocyanin Cyanidin Pelargonidin Delphinidin Forkmann, 1991 Development of Novel Flower Colour
- 26. • Anthocyanin (glycosides) are major contributing to flower colour (Stafford, 1990) • Phenylalanine is the precursor and synthesized by phenyl propanoid pathway (Springob et al, 2003; Tanaka and Mason, 2003) • The flower colour modification via genetic engineering is focused on metabolic engineering of the flavonoid pathway • Primary function of flavonoid pigments in flowers- Attract insects and other animals which help in crosspollination (Brouillard and Dangles, 1993) Provide protection against U.V radiation (Dixon et al, 1995)
- 27. CHS- chalcone synthase CHI- chalcone isomerase FLS-flavonol synthase F3'H- flavanone 3'-hydroxylase F3'5'H- flavanone 3' 5'- hydroxylase DFR- dihydroflavonol 4- reductase ANS- anthocyanidin synthase FNS- flavone synthase Anthocyanin biosynthetic pathway http://en.wikipedia.org/wiki/Anthocyanin
- 28. Antisense RNA technology Genetic engineering for white colour 1. Inhibition of chalcone synthase enzyme by Antisense RNA technology (in Petunia) Unstable duplex No translation Antisense CHSSense CHS Antisense CHS gene Sense CHS Antisense RNA technology
- 29. 2. DFR and F3H Silencing • When F3H is silenced in carnations transgenic plants were obtained with reduced anthocyanin and increased fragrance (Zuker et al, 2002) • Successful reduction of anthocyanin biosynthesis has been reported in Petunia (Krol et al, 1988) • Gerbera (Elomaa, 1993) • Chrysanthemum (Courtney-Gutterson et al, 1994) • Rose (Gutterson, 1995) • Carnation (Gutterson, 1995)
- 30. Petunia Cyanidin Delphinidin No pelargonidin due to substrate specificity of DFR Genetic engineering for orange and red colour(pelargonidin) A1 gene dihydroquercetin 4 reductase dihydrokaempferol (Meyer et al, 1987)
- 31. Pigment Crop Reference Enzymes Chalcones (yellow) Dianthus caryophyllus CHS, C2′GT Silencing CHI Aurones (bright yellow) Snapdragon, dahlia Aurone synthase 6′-deoxychalcones Cosmos, dahlia Davies and Schwinn (1997) CHS with CHR • Silencing of CHI Unstable chalcone acc. (Van blockland et al, 1993) No yellow pigment Petunia
- 32. • One of the aurone synthases, aureusidin synthase recently purified from yellow snapdragon petals (Nakayama et al, 2000) CHR chalcones, butein 3-O- glucoside and butein 4-O- glucoside C2' GT Stabilize chalcones (Ishida et al, 2003 and Okuhara et al, 2004) Petunia Carnation
- 33. Genetic engineering for blue colour • Rose, Chrysanthemum, and Carnations - no blue color - no delphinidin - lack of F3′5′H in their flowers • Blue carnations - Florigene, Australia (1996) F 3' 5' H Low delphinidin Petunia Carnation (Brugliera et al, 2000) Cytochrome b5 gene + F3'5' H
- 34. • Florigene's new lilac- and mauve-hued carnations- 'Moondust' and 'Moonglow' dominate the North and South American carnation cut- flower markets F3'5'H + DFR (Fukui et al, 2003)
- 35. • Blue rose- In 2004, Florigene gave mauve-lilac roses like 'BlueMoon' and 'Vol de Nuit' Delphinidin Petunia Delphinidin + cyanidin Red rose cv. Cardinal Dark burgandy not blue RNAi technology- BLOCK DFR Iris DFR Pansy delphinidin Three-gene package Blue Moon Tanaka et al, 2009
- 36. Colour modified flowers (A). Torenia hybrida cv. Summerwave Blue. Left; the host, middle; a transgenic line (co-suppressed DFR ) right; transgenic line (co-suppressed CHS) (Suzuki et al, 2000) (B) Lobelia erinus, Left: the host, right; a transgenic lobelia (lisianthus F3¢5¢H) (Kanno et al, 2003) (F) Orange petunia (pelargonidin made from red one producing cyanidin by down regulation of the F3¢H and expression of rose DFR) (Mizutani et al, 2003) (E) Torenia cv. Summerwave blue produced pink flowers (co-suppression F3¢5¢H) and darker pink flowers (over-expression F3¢H in pink flower) (Ueyama et al, 2002) (G) Yellow petunia (expressing Lotus japonica PKR)
- 37. • ABC Model was given to identify the floral organ identify gene (FOIG) • Gene A for sepal development (first outermost whorl) • Genes A and B together for petals in the second whorl • Genes B and C determine the stamens in the third whorl • Gene C alone specifies the carpel in the fourth whorl (Coen and Meyerowitz, 1991) Flower shape modification
- 38. • Antirrhinum majus B genes DEF and GLO in transgenic Torenia resulted in the conversion of sepals to petals • C gene from Rosa rugosa in Torenia resulted in a carpeloid structure in place of sepals (Kitahara et al, 2004) Homeotic genes Flower shape modification Arabidopsis- Agamous gene Antirrhinum- Deficiencies gene
- 39. Modification of Plant Architecture • Tobacco phytochrome B1 gene Chrysanthemum cv. ‘Iridon’ Shorter plants Larger branch angles (Zheng et al, 2001) • rolC Transgenic carnation (Cv. White Sim) increased axillary bud break 48% more stem cuttings/mother plant 3 times more flowering stems Stem cuttings from rolC plants exhibited better rooting (Zuker et al, 1999)
- 40. Floral Scent Modification • Secondary metabolites • Volatile, low-molecular-weight, give the flowers their unique, characteristic fragrances • Types of scent compounds : Class Precursor Types Examples Terpenoid isopentenyldiphosphate(IPP) and dimethylallyl diphosphate (DMAPP) Monoterpene Sesquiterpene Diterpene Geraniol, linalool Caryophyllene Phytol Phenylpropanoids (benzoids ) Aromatic amino acid (phenylalanine) (shikimate pathway) Eugenol, Methyleugenol Methylcinnmate Fatty acid derivatives Linoleic and linolenic acid Methyl jasmonate Jasmone Dudareva and Pichersky, 2006 www.sciencedirect.com
- 41. Genes responsible for scent production Flower crop Genes responsible floral volatiles Reference Clarkia breweri (S)-linalool synthase (LIS) gene Dudareva et al, 1996 Isoeugenol-O-methyl transferase (IEMT) Wang et al, 1997 Benzyl alcohol acetyl-transferase (BEAT) Dudareva et al, 1998 Salicylic acid carboxyl methyl transferase (SAMT) Ross et al, 1999 Benzoic acid carboxyl methyl transferase (BAMT) Pichersky and Dudareva, 2000 Benzyl alcohol benzyl transferase (BEBT) Dudareva et al, 2000 Petunia hybrida P. axillaris Benzoic acid/salicylic acid and carbonyl methyl transferase (BSMT) Negre et al, 2003 Benzyl alcohol /phenyl ethanol benzyl transferase (BPBT) Boatright et al, 2004
- 42. Rosa hybrida Germacrene D synthase Gueterman et al, 2002 Geraniol/citronellol acetyl transferase Shalit et al, 2003 Benzyl alcohol /phenyl ethanol benzyl transferase (BPBT) Boatright et al, 2004 Orcinol-O-methyl transferase (OOMT) R. chinensis Phloroglucinol-O-methyl transferase (POMT) Lavid et al, 2002 Antirrhinum majus (Snapdragon) Mycene synthase Ocimene synthase Dudareva et al, 2000 Stephanotis floribunda (Madagascar Jasmine) Salicylic acid carboxyl methyl transferase (SAMT) Pott et al, 2002 Arabidopsis thaliana (S)-linalool synthase (LIS) Caryophyllene methyl transferase (BSMT) Chen et al, 2003 Vanda Mimi Palmer Linalool synthase (LIS), acetyl-CoA acetyltransferase (ACA), 1- deoxy-D-xylulose 5-phosphate synthase (DXPS), 3-hydroxy-3-methylglutaryl- coenzyme A reductase (HMGR)
- 43. ODORANT1 regulates fragrance in petunia flowers cv. Mitchell • Petunia hybrida : volatile benzenoids ODORANT1 (ODO1) • Flowers fragrant in the evening and at night • Transcript levels of ODO1 before the onset of volatile emission decreased when volatile emission declined • ODO1 transgenic P. hybrida Mitchell benzenoid levels synthesis of precursors from shikimate pathway
- 44. Verdonk et al, 2005 Volatile benzenoids emission by Mitchell (M), RNAi lines (1,3,12,35)
- 45. PAP1 enhances phenylpropanoid and terpenoid production in Rosa hybrida cv. Pariser Charme • Arabidopsis PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) Rose • PAP1-transgenic rose lines phenylpropanoid (color and scent) when compared with control flowers (GUS) • PAP1- lines 6.5 times terpenoid (scent) Development of plant from somatic embryo i, ii, iii iv, v, vi vi, viii, ix GUS Transgenic control GUS Transgenic control following X-Gluc staining PAP1 Transgenic line
- 46. The levels of emission ((e), µg per flower per 24 h) and internal pools ((p), µg per flower) of volatile compounds produced by flowers of PAP1-transgenic lines 6, 11 and 12 when compared with control Zvi et al, 2012
- 47. Clarkia breweri benzyl alcohol acetyltransferase (BEAT) Eustoma grandiflorum BEAT catalyzing the synthesis benzyl acetate which constitutes up to 40% of C. breweri’s total scent output (Dudareva et al, 1998) No benzyl acetate No fragrance benzyl alcohol acetyltransferase (BEAT) Alcohol substrate 5-7 times higher levels of benzyl acetate Fragrant Aroma enhancement in transgenic Lisianthus using the Clarkia BEAT gene
- 48. Control and transformed adult flowering plantsLevel of benzyl acetate in control and transgenic lines after feeding with BA or water- (A) Leaf (B) Flower Aranovich et al, 2007
- 49. Linalool synthase (LIS) Linalool glucoside Linalool Lucker et al, 2001 Lavy et al, 2002 Strawberry alcohol acyltransferase (SAAT) Isoamyl alcohol Acetyl ester acc. Volatile unaltered Beekwilder et al, 2004 Alcohol acetyltransferase Acetate ester acc. Alcohol substrate Guterman et al, 2006
- 50. Genetic engineering for long vase life Ethylene is synthesized from the petals after full opening of the flower during senescence (Reid and Wu, 1992; Woodson et al,1992) Long vase life Senescence inhibition Inhibit ethylene biosynthesis Block ethylene action (Adams and Yang, 1979) aACS aACO
- 51. Strategies for long vase life 1. Antisense ACS and antisense ACO technology 2. Over expression of ACC deaminase (metabolises ACC before it converts to ethylene) 3. Over expression SAM hydrolase which converts ACC in other metabolite 4. Expression of gene for isopentenyl transferase (ipt) giving increased level of cytokinin
- 52. Antisense ACC synthase (aBoACS1) Antisense ACC oxidase (aBoACO1) Delayed senescence of Petunia flower transformed with antisense ACC synthase and oxidase genes Antisense BoACO1 gene is more efficient than antisense BoACS1 gene to reduce ethylene Ethylene production declines Ethylene production by leaf of cultured stem Huang et al, 2007
- 53. Antisense ACC oxidase delay carnation petal senescence Carnation cv. ‘Scania’ and ‘White Sim’ containing the antisense ACO gene and NPT II gene Character Control Transgenic Vase life 5 days 8-9 days Senescence Inrolling of petals Discoloration (normal) Transformed and control plant after 8 day of harvest
- 54. Ethylene production by control and transgenic lines Transgenic line Exogenous Ethylene Induce ACS and ACO Savin et al, 1995
- 55. Extended vase life of transgenic carnations using ipt gene • Ipt gene increased level of cytokinin • Transgenic carnations showed long vase life of 16 days in water. This is double the life of non-GM varieties (Kosugi et al, 2002)
- 56. Commercialization Hurdles - for GE Ornamental crops • Ornamentals have much smaller market value than food crops • High cost of analysis, risk assessment and regulatory approval - Super carnations, color-modified Torenia • Regulatory approval for field testing takes months or years • Molecular characterization require PCR-based identification test for which fee of 30,000 EURO Chandler, 2013
- 57. Marigold- French Vanilla Hybrid Burpee Seed Company., USA- creamy-white fully double flowers www.burpee.com
- 58. Heavily scented and boldly coloured tuberose Cv. Yellow Baby Sensation Cinderella Double Pink Super Gold Flower Single Single Single Double Single Flower colour Yellow Pink Lavender pink Pink Dark yellow Bulb size 10 cm 10 cm 10 cm 12 cm 12 cm Stem length 35 cm 45 cm 35 cm 65 cm 65 cm Ludwig & Co., Holland, 2013 www.floraculture.au
- 59. Conclusion GE is breeding tools that future generations can use to tackle environmental challenges No genetic barriers (gene from strawberry, maize) Novelty through genetic engineering (blue carnation and rose) Speed of improvement Altered plant byproduct, form and colour Creation of genetic variation