Genetic engineering in floriculture
- 1. GENETIC ENGINEERING IN FLORICULTURE Speaker : Zaman Mariya S. Course No. : MBB 591 Major Sub. : Plant Biotechnology Reg. No. : 04-1273-2010 ANAND AGRICULTURAL UNIVERSITY Major Guide : Dr. G. C. Jadeja Minor Guide : Dr. J. G. Talati Date :15 /09/2012 Time : 14:00 Hrs wel come
- 2. The Floriculture Industry………. Biotechnology In Floriculture Current Status Of Genetic Modification In Floriculture Transformation Color Modification Vase life Flower Scent Modified Plant Structure and Architecture Disease Resistance Case studies Conclusion Future Prospects 2
- 3. THE FLORICULTURE INDUSTRY • Floriculture is considered to include the cut flowers, potted plants, and ornamental bedding plants and garden plant industries (Chandler, 2003). • The worldwide production value of flowers and floricultural plants is approx. 50 billion EURO. • From the per capita consumption data provided by the Dutch flower council (Flower Council of Holland, 2007), it can be extrapolated that the global consumption value at consumer level is somewhere between 100 to 150 billion EURO. • The main areas of production and consumption of floricultural products are in the United States and Europe, with a significant industry in Japan. The area of production in China is also increasing rapidly. • The highest consumption per head is in the Netherlands, Luxembourg, Germany, Austria, and France. 3
- 4. Orchids Arachnis Aranda Aranthera Cattleya Cymbidium Dendrobium Lycaste Paphiodelphium Miltonia Odontoglossum Chrysanthemum Gerbera Antirrhinum Rose Carnation Cut flowers Bulbs and corms Gladiolus Tulips Lilies Tuberose Amaryllis Iris 4
- 5. World Flower Production and Consumption Production Consumption China Kenya Columbia Japan Europe USA $40 billion (Getu, 2009) Market grow 20% annually Japan EU USA 5
- 6. The global flower industry thrives on novelty. Genetic engineering is providing a valuable means of expanding the floriculture gene pool so promoting the generation of new commercial varieties. Engineered traits are valuable to either the consumer or the producer. At present, only consumer traits appear to provide a return capable of supporting what is still a relatively expensive molecular breeding tool. The goal of genetic engineering is to improve the characteristics of flowers such as, flower colour, vase life, floral scent, flower morphology, disease as well as pest resistance, flower productivity, timing and synchrony of flowering. Biotechnology In Floriculture 6
- 8. Genetic modification can be used to transfer new specific traits into the plant Conventional Breeding many gene and limited by genetic incompability Plant biotechnology single gene with no specific to plant species Genetic engineering: Manipulation of plant genome through recombinant DNA technology to alter plant characteristics. 8
- 9. Gene transfer methods Indirect Direct Most widely used More economical More efficient Transformation success is 80-85% Agrobacterium mediated gene transfer Particle bombardment or micro projectile Direct DNA delivery by Microinjection or PEG mediated uptake Ultrasonication Electroporation Chandler and Brugliera, 2011 9
- 10. Gene transformation Bacterium mixed with plant cells GENE identified and isolated Gene inserted into Ti plasmid Gene replication Gold particles coated with DNA Cells shot with gene gun and DNA incorporated into plant cell chromosome Ti plasmid moves into plant cell and inserts DNA into plant chromosome Cells screened for transgene Transformed cells selected with selectable marker Transgenic plant regenerated from single transformed cell 10
- 11. Color and color patterns Flower color: most important trait, dictating consumer attraction Role of color: • Attraction of pollinators • Function in photosynthesis • In human health as antioxidants and precursors of vitamin A • Protecting tissue against photooxidative damage • Resistant to biotic and abiotic stress • Symbiotic plant-microbe interaction • Act as intermediary for other compounds Color pattern: Differential accumulation of pigment(s) 11
- 12. Pigments Compound Types Compound Examples Typical Colors Porphyrins Chlorophyll Chlorophyll a & b Green Flavonoids Anthocyanins Pelargonidin, Cyanidin, Delphinidin, Peonidin Red, Blue, Violet Anthoxanthins Flavonols Kaempferol, Quercetin, Fisetin, Kaempferide, Morin, Myricetin, Myricitrin, Rutin Yellow Flavones Apigenin, Biacalein, Chrysin, Diosmetin, Flavone, Luteolin Yellow Isoflavones Diadzin, Genistein, Enterodiol, Coumestrol, Biochanin Flavonones Eriodictyol, Hesperidin, Naringin, Naringenin Colorless Co- pigments Flavans Biflavan, Catechin, Epicatechin Colorless Co- pigments Carotenoids Carotenes Lycopene, α-carotene, β-carotene, γ-carotene Yellow, Orange, Red Xanthophylls Lutein, Cryptoxanthin, Zeaxanthin, Neoxanthin, Rhodoxanthin, Violaxanthin, Canthaxanthin, Astaxanthin Betalains Betacyanins Reddish To Violet Betaxanthins Miraxanthin & Portulaxanthin Yellow To Orange 12
- 13. Chlorophylls and carotenoids are in chloroplasts Anthocyanins are in the vacuole 13
- 14. Factors on flower color perception pH of the vacuole: pH of the vacuole is acidic Small changes of pH have visible effects on flower color Metal ions: Metal complexing has a blueing effect on flower color Co-pigmentation: Co-pigmentation of anthocyanins with the colourless flavonols and flavones is an important factor influencing pigmentation. Co-pigmented flowers give a mauve colour, whereas in the absence of flavonols maroon flowers are formed. Co-occurrence of anthocyanins and yellow pigments Mixtures of Ans and yellow flavonoids were found in the orange- yellow or orange-red flowers of antirrhinum and bronze flower colour of helichrysum. Tanaka et al., 2009 low pH to high pH
- 15. Protein (Enzyme) Red Pigment Springob et al., 2003 Genes contain regulatory region and coding region Regulatory region Coding region The genes consist of DNA which is made up of four chemical letters The chromosome is made up of genes Chromosomes-23 pairs A cell 15
- 16. Genes involved in pigment synthesis Structural (enzyme) gene: Gene that codes for any RNA or protein product other than a regulatory protein. Enzyme Gen e Species CHS (Chalcone synthase) Chs Antirrhinum, Chrysanthemum, Orchid, Rosa, Dianthus CHI (Chalcone-flavanone isomerase) Chi Antirrhinum, Petunia, Eustoma, Dianthus F3H (Flavone 3-hydroxylase) F3h Antirrhinum, Calistephus, Chrysanthemum, Dianthus, Orchid F3’H (Flavonoid 3’ hydroxlase) F3’h Antirrhinum, Dianthus, Petunia F3’5’H (Flavonoid 3’,5’- hydroxlase) F3’5’h Calistephus, Eustoma, Petunia FLS (Flavonol synthase) Fls Petunia, Rosa FNS (Flavone synthase) FnsII Antirrhinum, Gerbera DFR (Dihydroflavonol-4- reductase) Dfr Antirrhinum, Calistephus, Gerbera, Orchid, Dianthus, Petunia Vainstein, 2004 Regulatory Genes Structural (Enzyme) Genes
- 17. Regulatory genes: Influence the type, intensity and pattern of flavonoid accumulation but do not encode flavonoid enzyme. Two classes of regulatory genes are identified: TF with MYB domain TF with MYC/bHLH motif (Vainstein, 2004) Plant Gene Myb Myc Petunia Rosea, mixta Delia Gerbera Gmyc I Perilla MybpI Petunia Phmyb3, An2, An4 An1 17
- 18. Genes involved in carotene pigment synthesis (Vainstein, 2004) Gene Enzyme Dxs Deoxyxylulose 5- phosphate synthase Dxr Deoxyxylulose 5- phosphate reducoisomerase Lpi LytB protein Gps Geranyl diphosphate synthase Fps Fernsyl diphosphate synthase Ggps Geranyl geranyl diphosphate synthase Psy Phytotene synthase Zds β-Carotene dessaturase Lcy-b Lycopene β-cyclase Lcy-c Lycopene β-cyclase Nsy Neoxanthin synthase Ccs Capsanthin capsorubin synthase Ptox Plastid terminal oxysidase 18
- 20. Colour modification done by: Over expression of structural genes Use of sense or antisense enzyme construct Inhibition of key biosynthetic enzyme Addition of an enzyme in a particular biosynthetic step 20
- 21. FLORIGENE scientists Isolated the blue gene in 1991 and patented in 1992. Petunia gene didn’t work in roses, so FLORIGENE scientists used their techniques on carnations— a much easier species to manipulate than roses. In 1996, Florigene developed mauve-coloured carnation, FLORIGENE Moondust and it was the world's first genetically modified flower on sale. In 1997, developed second genetically-modified carnation, FLORIGENE Moonshadow with a richer and true purple colour. Successfully developed a range of transgenic violet carnations by introduction of a F3′5′H gene together with a petunia DFR gene into a DFR-deficient white carnation. Fukui et al., 2003 mooniquemoonpearlmoonvelvetmoonberry 21
- 22. • Why a natural rose could not have the true blue colour? "Flavonoid 3', 5'- hydroxylase" is one of the key enzymes involved in the flavonoid biosynthesis for blue colour development deficient in rose. Natural rose did not have delphinidin. pH of cell sap is 4.0-4.5 (acidic). Cell sap is govern by 7 genes and each gene contributes 0.5 pH Anthocyanidin The price for a single blue rose is about $22 to $33. Source: The Japan Today The transgenic rose variety ‘‘Applause’’ was commercially released in Japan in 2009 (Tanaka et al., 2009) 22
- 23. Blue Gene Technology www.suntory.com and www.florigene.com.au In April of 2005, Suntory Ltd. and Florigene Ltd. announced the production of a blue rose by introducing three transformation constructs simultaneously into roses: To make a blue roses: (1) Turn off’ the rose DFR gene (2) Insert pansy gene to open the ‘blue’ door (3) Insert iris DFR gene to make blue pigment 23
- 24. Silencing anthocyanin biosynthetic pathway by a) Transcriptional down-regulation b) By inactivating the key enzymes CHS gene F3’5’H gene DFR gene More sensitive to ranges of stresses Alternative targets, better and increased fragrance Zuker et al., 200224
- 25. Gerbera (Courtney-Gutterson et al. 1994), Genetic engineering for white flower (Elomaa 1993), ChrysanthemumPetunia (Krol et al. 1988), Rose (Gutterson 1995), Carnation (Gutterson 1995). 25
- 26. Genetic engineering for red/ orange flowers •Cyanidin and delphinidin derivatives occur but no pelargonidin derivatives in petunia. •Petunia DFR cannot able to reduce dihydrokaempferol because it shows substrate specificity which lead to the synthesis of leucoanthocyanidins. •Over expression of A1 gene + abundant substrate –opens a new pathway leading to the synthesis of novel brick red colour petunia. Maize A1 gene encodes dihydroquercetin 4 reductase Meyer et al., 198726
- 27. Genetic engineering for yellow flowers • Aurones are bright yellow flavonoids found in species such as snapdragon, dahlia etc.. • Aurone synthase, more specifically aureusidin synthase (AS), was purified from yellow snapdragon petals and the cDNA encoding the enzyme was cloned. • A pale yellow petunia expressing a Lotus japonica PKR gene. 27 •Chalcone and aurones contribute to yellow colours • The most common chalcone, THC, is yellow but is spontaneously isomerized to naringenin in vitro and rapidly isomerized in vivo by CHI. • Discovery of chalcone 2′- glucosyltransferase (C2′GT) enzyme - stabilizes the chemically unstable chalcone. • Recent activity: (Okuhara et al. 2004) Lack of CHI activity + presence of UDP-glucose: THC 2’ glucosyltransferase (C2’GT) activity = yellow carnation
- 28. Plant species Original colours Gene sources Methods Produced flower colors References Cyclamen persicum Purple Endogenous F3’5’H Antisense Red to pink Boase et al. (2010) Gentiana sp. Blue Endogenous CHS Antisense White Nishihara et al. (2006) Blue Endogenous F3’5’H RNAi Lilac to purple blue Nakatsuka et al. (2008b) Nierembergia sp. Violet Endogenous F3’5’H Antisense Pale blue Uyema et al. (20006) Ostespermum hybrida Magenta Endogenous F3’5’H RNAi reddish Seitz et al. (2007) Gerbera hybrida DFR Over expression Petunia hybrida Blue Mazus psonilum CHS Dominant negative Pale blue Hanumappa et al. (2007) Red Lotus japonicus PKR Over expression Variegated red Shimada et al. (2006) Rosa hybrida Red to pale cyanic Viola sp. F3’5’H Over expression Bluish Katsumoto et al. (2007) Torenia hybrida Blue Endogenous ANS RNAi Whitish to pale blue Nakamura et al. (2006) Blue Endogenous ANS Antisense Pale blue Nakamura et al. (2006) Blue Antirrhinum majus AS Over expression yellow Ono et al. (2006) Flower colour modifications by regulating flavonoid biosynthesis 28
- 29. value of a cut flower =Post harvest longevity Senescence highly controlled process requires active gene expression & protein synthesis for programmed cell death. Increased respiration and ethylene production induction of catabolic enzymes resulting in decreased proteins Genetic engineering for longer vase life Aging of petals Inhibiting or by blocking perception of ethylene 29
- 30. Florigene has developed carnation flowers with enhanced vase life using antisense RNA technology. Down regulated petal ACC synthase Control STS Transgenic 30
- 31. A high level of resistance in osmotin, pr-1 and/or chitinase genes to a major carnation pathogen (fusarium oxysporum f. Sp. Dianthi ) was demonstrated in greenhouse tests. (Zuker et al., 2005) Genetic Engineering for biotic stress Transformation of chrysanthemum cultivar 'shuho- no-chikara' was modified by delta-endotoxin gene cry1ab (mcbt) from bacillus thuringiensis biological activity against lepidopteran insects into chrysanthemum. Protection of flower crops against coat protein viruses (William R. Woodson 1991) Transgenic chrysanthemum showing resistance against chrysanthemum stunt viroid (csvd) and TSWV. 31
- 32. Genetic engineering for improved shape, size ABC MODEL 32 The combination of three genes that give rise to the flower parts. A sepals A + B petals B + C stamens C carpels • The ABC model (Coen and Meyerowitz 1991) and its modified version (Theißen 2001) are known to be applicable to a broad range of plants (Kim et al. 2005).
- 33. 33 Genetic engineering for improved shape, size Constitutive expression of Antirrhinum majus B genes DEF And GLO in transgenic torenia resulted in the conversion of sepals to petals. Constitutive expression of the C gene from Rosa rugosa in torenia resulted in a carpeloid structure in place of sepals (Kitahara et al. 2004) Studies on homeotic mutants have clarified many important aspects of genetic control on flower development. Deficiencies genes and AGAMOUS genes isolated from Antirrhinum majus increased interest in novel flower shapes through molecular manipulation. Transformant Wild type
- 34. Genetic engineering for floral scent may enhance the value of cut flowers to consumers… Fragrance numerous volatile aromatic organic substances present in the flower. Such as, hydrocarbons, alcohols, aldehydes, ketones, esters, ethers Manipulation of fragrance in flowers chemicals contributing to the fragrance of roses, their pathways of synthesis and enzymes controlling these pathways to be identified.34
- 35. Genetic engineering to modify plant architecture Control of plant height is of great importance in floriculture 35 • A petunia plant transformed with Arabidopsis gai-1 (right). • Genetic modification may replace chemical growth retardants in future.
- 36. Limitation of Plant gene transformation • Improvement of traits controlled by many gene. • Improvement of traits controlled by gene that has not been identified. • Requires high technology knowledge and equipment. • Expensive cost. • Government regulation. 36
- 37. o Flower colour changed from purple to almost white by the down-regulation of the CHS gene Surfinia Purple Mini Tsuda et al., 2004 Surfinia Pure White 37Japan 1 Case studies
- 38. Flowers of transgenic Surfinia Purple Mini plant harboring antisense DFR gene Expression of DFR gene change the expression of the flavonol synthase and flavone synthase gene Contd….. C 38Japan Tsuda et al., 2004
- 39. Vector construction Figure 2. Schematic representation of the expression cassettes in T-DNA of binary vectors constructed for colour modification. 39Japan
- 40. Generation of orange petunia expression of rose DFR gene + Suppression of F3H gene by antisense or RNAi method Generation of red petunia expression of rose DFR gene T-DNA copy number analysis of transgenic plants show that three most stable lines, two plants had a single copy insert, and the other one had two. Contd….. 40Japan Tsuda et al., 2004
- 41. Modification of co-pigment by Suppressing flavonol biosynthesis resulted in darker and slightly redder colour. A flower of a transgenic plant expressing rose FLS gene has a paler flower Surfinia Violet Mini transgenic petunia expressing torenia FNSII gene Creeping character did not change 41Japan Tsuda et al., 2004
- 42. Engineering of the Rose Flavonoid Biosynthetic Pathway Successfully Generated Blue-Hued Flowers Accumulating Delphinidin Katsumoto et. al (2007)Japan o Rosa hybrida lacks violet to blue colour due to the absence of flavonoid 3’,5’-hydoxylase (F3’5’H) enzyme which produces delphinidin-based anthocyanins. o Other factors such as co-pigments and vacuolar pH also affect flower colour. o Expression of the viola F3’5’H gene accumulates (~95% high) delphinidin a novel bluish flower colour. 42 2
- 43. • For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, the endogenous dihydroflavonol 4-reductase (DFR) gene was down- regulated and overexpressed the Iris3hollandica DFR gene in addition to the viola F3’5’H gene in a rose cultivar. • The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. • Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations. 43 Katsumoto et. al (2007)Japan
- 45. WKS77 WKS82 WKS100 WKS116 WKS124 WKS140 Rose Varieties transformed with pSPB130 and their flower colour changed are shown Schematic representation of T-DNA region of binary vectors constructed for colour modification for the constitutive over expression of the viola F3’5’H BP40 gene and the torenia 5AT gene in rose. 45
- 46. o Correlation of delphinidin content and petal colours in transgenic Lavande. o Pure red and blue have hue values of 360° and 290° in the hue angle. 46 Japan Katsumoto et. al (2007)
- 47. Host flower violet-coloured transgenic flower 98% delphinidin 98% delphinidin Madam Violet Seiryu The vector pSPB919 is to down-regulate the endogenous rose DFR gene using RNA interference (RNAi) and to over express the iris DFR and the viola F3’5’H genes.
- 48. • Northern blot analysis of LA/919-4-10. • The expected sizes of the transcripts of viola F3’5’H BP40 (1.8 kb) and iris DFR (1.7 kb) genes & smaller size was detected for rose DFR mRNA (A). • A rose DFR probe detected about 23 bp small sized RNA, which was supposed to be a degraded endogenous rose DFR transcript with RNAi (B). • Delphinidin contents was confirmed in all transgenic (KmR) progeny of LA/919-4-10. • The flowers of F1 and F2 progeny contained exclusively delphinidin. 48 (si RNA)
- 49. Nakatsuka et.al (2009)Japan 49 (A)A binary vector, pSMABR-rolCpro-intF3’5’H:5/3’ATir (B)The typical flower of wild-type gentian cv. ‘Albireo’ (C) 5/3’AT – suppressed transgenic gentian clone no.1. (D) 5/3’ATandF3’5’H double suppressed transgenic gentian clone no.15. 3
- 50. •Expressions of anthocyanin biosynthetic genes in transgenic gentian plants. •The membranes were hybridised with DIG-labelled probes for GtCHS, GtF3’5’H and 5/3’AT, respectively. •WT indicates untransformed wild-type gentian cv. ‘Albireo’. Ethidium bromide-stained ribosomal RNA bands (rRNA) are shown as a control. •Accumulation of anthocyanidin and flavone derivatives in transgenic gentian plants. (B) Flavone conc. in the petal of an untransformed control plant (WT) and transgenic plants were determined by measuring the absorbance at 330 nm using HPLC analysis with apigenin and luteolin as the standards. • Data are the average ±SD of five replicate flowers. (A) Anthocyanidin conc. in the petal of an untransformed control (WT) and transgenic plants were determined. • Data are the average ±SD of five replicate flowers. 50 RNA gel blot analysis
- 51. Mercuri et. al (2001)Italy GFP detection in Eustoma (Lisianthus) flower petals. (A) GFP detection in Osteospermum ligules flower petals. (A) GFP = Green Fluorescent Protein 51 4
- 52. Block schemes of the gfp-expression constructs used in this work.
- 53. GFP detection in leaves and roots of transgenic plants. (A, B) Leaf trichomes from Osteospermum transfected with vector alone. (C, D) Leaf trichomes from Osteospermum transfected with mGFP5. (E, F) Roots from Osteospermum transfected with vector alone (left) or mGFP5 (right). (A, C, E) were illuminated with white light; (B, D, F) were illuminated with UV light.53 GFP detection in flower and leaf extracts PAGE analysis of plant extracts Western blot analysis A B C D E F
- 54. (A) Phenotypes of transgenic Torenia flowers. • (Upper) Flower color under white light. • (Lower) Cellular fluorescence from adaxial side of petal in each line. (B) Expression analysis of each transgenic line by RT-PCR/Southern blotting Coexpression of the Am4CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). 54Japan Ono et. al (2006) A B 5
- 56. Production of picotee-type flowers in Japanese gentian by CRES-T A B Wild type flower‐Solid color suppression of pigment production generates picotee type flower Nakatsuka et al., 2011Japan CRES-TChimeric repressor gene-silencing technology 56 6
- 57. • (CRES-T) is an efficient gene suppression system which worked successfully in Japanese gentian. • A chimeric repressor of the anthocyanin biosynthetic regulator gene GtMYB3, under the control of the Arabidopsis actin2 promoter, was introduced into blue- flowered gentian. • Of 12 transgenic lines, 2 exhibited a picotee flower phenotype with a lack of pigmentation in the lower part of the petal. • HPLC analysis showed that the petals of these lines contained less anthocyanin and more flavone than the wild-type. • The expressions of ‘late’ flavonoid biosynthetic genes, including F3H, F35H, DFR and ANS, were strongly suppressed in petals of these transgenic plants. 57
- 58. • Expression of flavonoid biosynthetic genes in transgenic gentian plants. • The expression levels of GtMYB3-SRDX and endogenous flavonoid biosynthetic genes were determined by semi- quantitative RT-PCR analysis in wild-type and GtMYB3- SRDX expressed transgenic gentian clone nos. 7 & 11 • Schematic representation of pSMABR-AtACT2pro- GtMYB3-SRDX. • Bar herbicide bialaphos resistance gene as a selectable marker. • NOSp promoter of nopaline synthase (NOS) gene from A. tumefaciens. • rbcSt terminator of RuBisCO small subunit 2B gene from Arabidopsis. • NOSt terminator of NOS gene. • LB left border; RB right border. semi-quantitative RT-PCR analysis 58
- 59. Flavonoid analysis in the flowers of transgenic gentian plants by HPLC A & D- wild type B & E- transgenic gentian clones no. 7 C & F- transgenic gentian clones no. 11 Anthocyanins Flavones 59
- 60. Ethylene‐Insensitive Transgenic Petunias Jones et al, 2005USA Senescence was delayed by approx. 8 days 60 7 Ethylene sensitive (MD) Ethylene-insensitive (35S:etr1-1)
- 61. Nine genes encoding putative cysteine proteases Protein content (A) and protease activity (B) in wild-type Petunia (MD) & 35S:etr1- 1 transgenic (etr-44568) petunia corollas during flower development. PhCP2 to PhCP10 61 Jones et al, 2005USA • In this study, 35S:etr1-1 transgenic petunias have been used to see how ethylene regulates flower senescence. • To compare the senescence programmes in ethylene-sensitive (MD) and ethylene-insensitive (etr- 44568) flowers, a comparative analysis was conducted of age- related changes in total protein, protease activity, and the expression of nine cysteine protease genes in the petals of MD and etr-44568 petunias.
- 62. ETR1-1 - Makes plants ethylene insensitive Long Lasting Flowers Etr1-1 62 Control Etr1-1Control Etr1-1
- 63. Generation of phenylpropanoid pathway-derived volatiles in transgenic plants • Rose alcohol acetyltransferase produces phenylethyl acetate and benzyl acetate in petunia flowers. • Esters are important volatiles contributing to the aroma of numerous flowers and fruits. • Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). • To study the function of rose alcohol acetyltransferase (RhAAT), they generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. • Phenylethyl alcohol and benzyl alcohol produce the corresponding acetate esters, not generated by control flowers. • The level of benzyl acetate is three times more than phenylethyl acetate in different transgenic lines of petunia. USA Pichersky et al.,200663 8
- 64. Molecular analyses of transgenic petunia plants expressing RhAAT RNA blot GC–MS analysis of volatile compounds benzyl acetate phenylethyl acetate 64 USA Pichersky et al.,2006
- 65. Transgenic rose lines harboring an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced resistance to powdery mildew • An antimicrobial protein gene, Ace-AMP1, was introduced into Rosa hybrida via Agrobacterium-mediated transformation. • PCR analysis for both Ace-AMP1 and neomycin phosphotransferase (npt II) genes, showed that 62% of these plants were positive for both transgenes. • These lines were further confirmed for stable integration of Ace-AMP1 and npt II genes by Southern blotting. • Transcription of the Ace-AMP1 transgene in various transgenic rose lines was determined using Northern blotting. USA Li et al., (2003)65 Fig. a) Powdery mildew development on leaflets & b) on whole leaves Control Control Transgenic Transgenic 9
- 66. 66 PCR analysis of transgenic plants of Rosa hybrida cv. Carefree Beauty Tnos Ace-AMP1 Penh-35S Pnos Npt II Tg7 EcoR 1 The binary vector pFAJ3033. The Ace-AMP1 gene is driven by CaMV 35S with a duplicate enhancer region, and is terminated by (NOS). Southern blot hybridization analysis of transgenic and control rose plants. A) Probed with a 0.35- kb fragment of the Ace-AMP1gene. B) Probed with a 0.7-kb fragment of npt II gene Northern blot analysis whereby a 0.35-kb fragment of the Ace-AMP1 gene was used as a probe A) B)
- 67. Conclusions • Genetic engineering overcomes almost all the limitations of traditional breeding approaches. • Recent developments in plant molecular biology provide opportunities to use techniques of genetic engineering for improvement of flower crops for modify flower colour, improve vase life, floral morphology, scent and disease resistance. • Spectral difference in flower colour is mainly determined by the ratio of different classes of pigments and other factors and knowledge of flower coloration at the biochemical and molecular level has made it possible to develop novel color. 67
- 68. • Recently genetic engineering has demonstrated the best examples such as ‘Moon’ series of transgenic carnations and transgenic blue rose marketed in North America, Australia and Japan. • Ethylene sensitivity regulates the timing of flower senescence and incorporation of etr1-1 gene delayed senescence in petunia flowers. • Recent advances in the isolation of scent biosynthetic genes have provided the basis and created the opportunity for the biotechnological manipulation of floral scent. • Ace-AMP1 is a good candidate gene for genetic improvement of disease resistance in roses. 68 Conclusions
- 69. Future prospects and new avenues 69 • New genes should be isolated that will have utility in floriculture, and new transformation methods for flower crops should be further optimized. • A genetic map of rose, which is commercially the most valuable cut flower, has now been developed. Identification of quantitative trait locus (QTL) from this map, in conjunction with genetic modification, will assist breeders to improve productivity, disease and insect resistance. • Information on expression of regulatory genes encoding transcription factors should be generated which have effects on flower colour, fragrance and disease resistance. • The manipulation of colour in the yellow and orange range will become increasingly feasible as more is learnt about the carotenoid biosynthesis pathway. • More research efforts are needed to modify flower shape
- 70. 70 Thank you
Editor's Notes
- #7: Traditionally, Breeding of ornamental plants has been based mainly on continuous crossing & selection, Several decades have combined for commercially important characteristics into an elite genotypes.
- #12: They show antibacterial, antifungal, antitumor and anti-inflammatory properties.
- #15: A higher vacuolar pH (around 4.0 rather than3.0) is necessary for the blue flower colours Fe3+, one Mg2+ and two Ca2+ ions Anthocyanins are a red colour under low pH environments and a blue colour under neutral or alkaline pH environments Vacuolar pH is generally regulated by vacuolar ATPase and pyrophosphase in plant cells. Anthocyanins can be used as pH indicators because their color changes with pH. Anthocyanins are pink in acidic solutions (pH < 7), purple in neutral solutions (pH ~ 7), greenish yellow in alkaline solutions (pH > 7), and colourless in very alkaline solutions where the pigment is completely reduced.
- #22: The development of the blue carnation was not the primary goal of the research team; no, They wanted to make a blue rose but, used
- #24: stopping the production of red pigment, allowing the production of blue pigment. They were able to stop the production of the red pigment by silencing dihydroflavonol reductase (DFR), the red producing gene. a delphindin gene from a pansy was inserted to allow blue pigment production. Finally, another DFR gene, this time from an iris, was inserted, to actually produce the blue pigment.
- #35: Metabolic engineering is a form of genetic engineering aimed at changing the way living things metabolize, or rearrange the nutrients they take in into different chemicals and thus make useful fragrances.
- #38: Petunia flower colours are mainly due to flavonoids. Flower colour modified by the suppression of flavonoid biosynthetic genes
- #39: They showed various phenotypes.
- #47: The percentage of delphinidin in the petals of individual transgenic plants was plotted against the flower colour represented by the hue value in degrees (hue angle).
- #51: For each plant, 5 mg total RNA was subjected to RNA gel blot analysis.
- #53: CaMV35S: Cauliflower Mosaic Virus 35S promoter. E35S: enhanced CaMV35S promoter. Nos-ter: nopaline synthase terminator. Signal peptide: signal sequence of the basic chitinase of A. thaliana. Hsp70 intron: maize heat-shock protein 70 intron. ST-LS1 intron: intron of the potato ST-LS1 gene. GmHsp 17.9 leader: soybean heat-heat-shock protein 17.9 gene leader sequence. 2xE35S-pro: CaMV35S promoter with a duplicated transcriptional enhancer. pA 35S: CaMV35S poly adenylation signal. The translation start (ATG) and stop codons (TAA and TGA), the chromophore aminoacid mutations (V163A, I167T, S175G, S65T and S65C), and the HDEL endoplasmic reticulum retention signal are indicated.
- #55: Phenotypic and expression analyses of transgenic Torenia Flowers