Mutation and isolation of chimeras through in vitro approaches in chry

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Techniques of isolation of novel mutants

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Speaker: Arvind Kumar Verma M.Sc . Horticulture IARI, New Delhi Chairperson: Dr. K.V. Prasad Mutation and isolation of chimeras through in vitro approaches in chrysanthemum

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In the international cut flower market it ranks 2 nd and in Japan and China it is the number one flower crop. Classical breeding has limitations: restricted gene pool self-incompatibility longer ray florets incompatibility due to parental ploidy differences Out of 2323 mutant varieties reported in IAEA database nearly 1/10 th (233) of them belong to chrysanthemum. Introduction

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Main bottleneck of mutation induction in chrysanthemum is the development of partial chimeras therefore, a large number of new flower colour mutants are lost every year which can’t be isolated through conventional propagation techniques. It is possible to isolate a portion of a branch or an entire branch if it is completely mutated, but it is difficult to isolate such mutants/chimeras which are often limited in extent and may only expressed in flower. Mutation breeding coupled with in vitro regeneration is a useful approach to establish novel mutants in pure form and facilitate production of a wide range of new coloured cultivars. Contd……

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What is Chimera Plant composed of two genetically different types of tissues

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Historical Background The term chimera comes from Greek mythology. It is a composite beast with the head of a lion, body of a ram and the tail of a snake Charles Darwin describes many types of plant variations in his book “The Variation of Animals and Plants under Cultivation” in 1868 In 1907, Hans Winkler demonstrated how a graft chimera is developed. He grafted two dissimilar plants (nightshade on tomato) and after the union healed he cut through the graft union Erwin Baur determined that variegated plants that show green and white leaves are naturally occurring chimeras. He worked with a geranium called “Freak of Nature” in 1909 Satina and Blakeslee described the tunica of the dicot shoot meristem having distinct layers in 1941

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Types of chimeras Periclinal A periclinal chimeras in which one or more entire cell layer(s) genetically distinct from another cell layer(s). It is a common cause of leaf variegation. Mericlinal In a mericlinal chimeras the mutant cells are only in the part of a cell layer. It is usually thought of as a transition as cells divide to form a periclinal chimeras. Sectorial Sectorial chimeras have a sector of all cell layers that is genetically different. It is an unusual chimeras and it would revert to a periclinal chimeras or an all mutant or non-mutant meristem . Datta et al., 2005 Three kinds of chimeras based on the spatial arrangement of the genetically distinct cells within these cell layers

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Cells in each layer give rise to different tissues in the plant. The apex is organized into a layered region called tunica A region where layering is not evident called corpus L-I is outer most layer of cells form epidermis L-II is the next inner layer of cells and is responsible for the cortex. L-III is the innermost layer of cells and form pith Different layer of the apical meristem Garry et al., 2002

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Causes of chimera Chimeras have originated spontaneously due to mutation in one of the layers in the apical meristem. A chimera is possible because the plant's apical meristem is made up of relatively independent layers. Gene mutation Chromosomal mutation Cytoplasmic mutation Mutation occurring in the biochemical pathway

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Other sources of variegation Pattern variegation: It is a genetic trait of the cultivar and pattern is due to different cells in a tissue expressing colour genes. Transposons : It is a movable genetic element and randomly move about the chromosome creating genetic mosaic. It can be distinguished from chimeras because the variegation pattern is inherited from seed. Pathogen infection: A variegation was found in Tulip flower that caused by a viral infection in the Netherlands. This was caused tulip breaks and was the cause for tulipomania Olbricht et al., 2006

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Various mutagens used in mutation breeding of ornamental plants Park and Song, 2005

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Number of mutation derived varieties in the world Park and Song, 2005

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Numbers of ornamental mutants in the world Park and Song, 2005

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Mutagens used for the induction of mutation in chrysanthemum in the world Park and Song, 2005 Rank Country No. Of variety Mutagen (no. of variety) 1 Netherlands 80 γ- rays (68), x-rays (12) 2 India 46 γ- rays (45), Colchicine (1) 3 Germany 34 γ-rays (13), x-rays (22) 4 China 21 γ- rays 5 Russian Federation 17 γ-rays 6 Japan 14 γ-rays (8), chronic (6) 7 Belgium 7 x-rays 8 Poland 6 x-rays (3), γ-rays (3) Others 8 γ-rays, in vitro, x-rays Total 233

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Induction of mutation

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Effect of irradiation on survival of chrysanthemum plants Siranut et al., 2000

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Success story at IARI Radiation Dose Effect 10 Gy Hormesis 15 Gy 60-70% Survival (LD 30-40) 20 Gy 50 % survival (LD 50) 30 Gy Mortality Varieties Ajay 10 Gy 15 Gy 20 Gy 30 Gy Survival rate of plants at different doses of gamma rays Prasad et at., 2008

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Radiation Dose (Gy) Mutation frequency (%) Carbon ion 2 17.4 (37/213) Helium ion 5 12.8 (16/125) Helium ion 10 28.8 (32/111) Hiroyasu et al., 2008 Effect of different ion beam on survival of chrysanthemum plants cv. ‘ Taihei’

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Mahure, 2004 Effect of different EMS concentration on extent of abnormality (%) in chrysanthemum varieties Varieties EMS Conc. (%) 0.05 0.75 0.1 Red Gold 15.00 57.00 92.55 Yellow Gold 19.75 54.54 90.45 Karnool 30.65 81.56 96.49 Geetanjali 23.00 52.74 84.95 Aprajita 17.99 62.02 85.48 Shyamal 17.08 50.19 93.09

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Mahure, 2004 Effect of different doses of gamma rays on survival (%) of un-rooted shoot cuttings of chrysanthemum Varieties Doses of gamma rays ( Gy ) 10 15 20 Red Gold 92.85 75.55 31.75 Yellow Gold 80.65 42.17 14.07 Karnool 79.08 41.15 12.02 Geetanjali 95.11 67.72 10.14 Aprajita 90.56 65.17 10.25 Shyamal 75.98 50.55 20.00

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Phenotypic expression of chimeras a c e d b Two types of flower in a single branch Datta et al. , 2001

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In vitro techniques for isolation of chimeras

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Why in vitro isolation of chimeras ? It is not possible to isolate promising novel mutants through vegetative propagation because it is impossible to raise plants from petal and leaves in field condition It is possible to make multiple copies of the novel mutants through in vitro tools Sectorial chimeras can be isolate only by in vitro method and can not be isolated in field conditions as it is the main bottleneck of mutation breeding Easy to propagate without any seasonal barrier to raise large number of plantlets

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Effect of explants size on callus induction, shoot initiation and No. of shoots per explants Nahid et al., 2007 Size of Explant Callus Induction (%) Shoot Regeneration (%) No. of shoots per explants 3 mm 41.00 0.66 0.05 5 mm 63.00 55.66 0.18 7 mm 96.00 83.33 1.14 1 cm 38.66 3.33 0.05

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Isolation of chimera from ray florets of chrysanthemum Mishra at al., 2004 C D F E G H Lalima A Yellow colour with tubular petal and yellow colour with spoon shaped petal B

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Isolation of chimera from stem segments Mandal et al., 2000 M1 M2

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Ajay chimeras Isolation of chimeras in chrysanthemum cv. Ajay Prasad et at., 2008 Pusa Anmol A success story at IARI………………… M1 M2

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Effect of growth regulators for in vitro isolation of chimeras

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Nahid et al., 2007 Effect of growth regulators on callus induction and shoot regeneration (ml/l) (ml/l) (ml/l)

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Effect of growth regulators on direct shoot regeneration from chimeric ray florets of Chrysanthemum morifolium cv. ‘ Purnima ’ Mandal et al., 2000

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Effect of plant growth regulators on direct shoot organogenesis from chimeric ray florets of Dendranthema grandiflorum cv. `Puja' * = Significant, p= 0.01 Datta et al ., 2004

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Effect of growth regulators on axillary shoot multiplication from nodal explant Mandal et al., 2000

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Efficiency of solid-colour mutant recovery from different explants of chrysanthemum cv. Maghi Mandal et al., 2000

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Conclusion Large number of new flower colour mutants are lost every year due to formation of chimeras which can’t be isolated through conventional propagation techniques Main bottleneck of mutation induction in chrysanthemum is the development of chimera, therefore in vitro isolation of chimeras is the only viable option to isolate such novel mutants Mostly gamma rays is used to induced mutation in chrysanthemum while, chemical mutagens produced abnormalities Advantage of mutation breeding over conventional breeding techniques is the relatively short time required to produce a novel colour mutants if reliable techniques are available Appropriate combination of growth regulators are required for in vitro isolation of chimera

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Future Thrusts The combination of mutation breeding and in vitro culture tools open up new avenue for isolating mutants which can’t be otherwise isolated through conventional means Induction of a spectrum of colours open up avenue for large scale production of nutraceuticals pigments Characterization of genes involved in pigment biosynthesis can be easily performed to explore the possibilities of transferring them across varieties, species and genus In order to seek protection for isolated novel mutants, there is an urgent need to establish the DUS status for these mutants

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