Advances in Breeding Techniques of Bulbous Ornamental Crops

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Available online at www.ijpab.com

DOI: http://dx.doi.org/10.18782/2320-7051.7427 ISSN: 2320 – 7051 Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) Review Article

Advances in Breeding Techniques of Bulbous ornamental Crops

Neha Dogra* and K. K. Dhatt Department of Floriculture and Landscaping, Punjab Agricultural University, Ludhiana (Punjab) – 141004 *Corresponding Author E-mail: [email protected] Received: 5.03.2019 | Revised: 7.04.2019 | Accepted: 13.04.2019

ABSTRACT Bulbous crops are specialized modified underground stem structures in which food material is stored to overcome the unfavorable environmental conditions. These plants are prized for their magnificent flowers and for being grown for mass effect. Breeding techniques are of two types i.e. conventional (Introduction, Selection, Hybridization, Mutation) and non- conventional (Genetic engineering, Biotechnology). Hybridization is the crossing of two or more plants which are genetically different from each other to produce a new crop. It is used to create genetical variation and to exploit the hybrid vigour. Mutation breeding is the sudden heritable changes occurred in an organism. Mutation may be caused by spontaneous and induced and the result of a change in the gene or chromosome. Anther culture is the process of using anthers to culture haploid plantlets. The technique was discovered in 1964 by Guha and Maheshwari. Embryo culture is to separation or isolation of plant embryo and growing to this in culture medium under controlled condition or lab. Genetic engineering is the artificial transfer of gene(s) from one organism to another to produce novel traits in the recipient living organism. It is of two types Direct or Indirect. Direct gene transfer are Chemical Mediated Gene Transfer, Microinjection, Electroporation, ParticlGun/Particle Bombardment, Liposome Mediated Gene Transfer Or

Lipofection and Indirect gene transfer are Agrobacterium tumefacians, Agrobacterium

rhizogenes. Molecular markers are classified on the based of basic stragety i.e. PCR and Non

PCR based. PCR based are (RAPD, AFLP, SSR, ISSR), Non PCR based (RFLP), Marker assisted

selection (MAS) is the selection of plants with desirable traits based on their molecular

genotype. It can be used alone or in combination with the classical methods of selection.

Key words: PCR, Mutation, Bulbous, Microinjection, Electroporation,

INTRODUCTION overcome the unfavorable environmental Bulbous flowering plants are prized for their conditions1. Gladioli (9500), Narcissus magnificent flowers grown for mass effect and (7000ha), Lilium (5000ha), Iris (2000ha), bulb are defined modified underground stem Tulips (1600ha) are the top five genera of structures in which food material is stored to bulbous plants acerage wise in the world.

Cite this article: Dogra, N. and Dhatt, K.K., Advances in Breeding Techniques of Bulbous ornamental Crops, Int. J. Pure App. Biosci. 7(2): 279-291 (2019). doi: http://dx.doi.org/10.18782/2320-7051.7427

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Out of total world floriculture trade (70, Selection: are the oldest breeding method and 34,922.88MT) bulbous ornamental trade the basis of all crop improvements. It is the contribute 4.46 % share quantity wise and 5.55 choosing the best out of one‟s crop continued % value wise However, in India total area over generations for development and under bulbous crops are 3500ha and gladiolus retention of already developed varieties. contributes maximum area (1200ha) followed Hybridization: is the crossing of two or more by tuberose (800ha). Kalimpong and Sikkim plants which are genetically different from contributing 30-35% of total bulb export. each other to produce a new genotype. It is Other areas are Srinagar (J&K) and Katrain, effective to combine all the good characters in Kullu (HP). Gladiolus, Tulip, Lilium, Dahlia, a single variety to create genetical variation Alstroemeria and Tuberose are the and to exploit the hybrid vigour. There are five commercially high valued bulbous crops. methods of hybridization based on relationship These crops get high prices in the market only between parental plants i.e. intravarietal, when they are healthy and appeal to intervarietal, interspeific, intergeneric and consumers so these objectives can only met by introgressive hybridization. By using these using different breeding techniques. hybridization techniques different hybrids has Breeding techniques are of two types been develop. i.e. conventional (Introduction, Selection, Role of hybridization in bulbous Hybridization and Mutation) and non- ornamental crops conventional (Genetic engineering, 1) Gladiolus: - started by William Biotechnology). Herbert in England. Colville hybrid was first Conventional Techniques:- commercial hybrid developed by James Introduction: is the process of introducing Colville in 1823 in England. Other hybrids are plants from their growing to a new locality. Gandavensis which is cross between Gladiolus The main objectives of introducing plant psittacinus x G. cardinalis. Nanus hybrid cross material from outside are for use as medicinal between G. cardinalis x G. venustus and or industrial purpose, to study origin and Glandanthera hybrid cross between Filigree x evolution of crop plants and in case of Acidanthere bicolor var. murielae. The ornamentals to enrich and fulfill the aesthetic inflorescence of gladiolus is spike and flowers values of gardens and for genetical are bisexual, trimerous florets. Protoandry and improvement of economic crops through direct distyly are occurring. Anthers mature with release, selection, as breeding materials. opening of flowers. Stigma receptive after Organizations involved in plant introduction three days of anthesis. In gladiolus are FRI (forest research institute, Dehradun), hybridization was done by following ways :- Botanical Survey of India, NPBGR.

Hybridization in Gladiolus

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Rao and janakiram got fragrant hybrid by Integrated Techniques for Overcoming crossing between Gladiolus dalenii x G. Pre- and Post-fertilisation Barriers callianthus and also reported that interspecific Various combinations of in vitro hybrid with mild fragrance were produced by pollination (cut-style and grafted-style crossing Gladiolus grandiflorus and G. method) and embryo rescue (ovary, ovule and callianthus. embryo culture, placental pollination). 2). Lilium:- the inflorescence of lilium is 3) Dahlia :- The inflorescence of dahlia terminal raceme or solitary perianth are 6 and is head or capitulum. Dahlia is a sepals and petals are 3, flowers are erect, bisexual flower. spreading, stamens are 6, ovary is superior and Both ray and disc florets of capitates stigma three lobed. Batistero (LA), La Reina inflorescence open in succession. The opening (LA), Boogie Woogie (OT) are fragrant starts from outer petals. In dahlia ray florets hybrids. Easter moon (Disease resistance), are female flower contain only stigma in the Pisa (Golden yellow cultivars), Yellito, China inner base of petals. While disc florets contain (pollen less cultivars). Loffler et al.9, crossed both female and male organs which complete fusarium resistant species Lilium dauricum flowers. (resistant) and Lilium longiflorum var. Gelria Hybrids of Dahlia3: (susceptible) got resistant hybrid.  Swami Vinayananda (Breeder): Breeding problems in lilium: Bhikkus Mother, Bhikkus Vivek, 1. Self incompatibility (failure of viable Jyotsana Lord Buddha, Prabodh, pollen to fertilize the flower), a major Sarada Devi, Swami problem, is of two types:- Gauriswarananda, Swamji. a) Pre fertilization barriers: – when  C. Sen and A.Sen (Breeder): Basu growth of pollen tube inhibited either dev and Jayanti at base of stigma or in style.  L.N. Singh Thakur (Breeder): b) Post fertilization barriers: – Chitchor fertilization occurs but embryo gets  R. Mitra (Breeder): Manjushri degenerate at later stage of  S.C. Dey(Breeder): Disco and development. Swami vinayananda 2. F1 hybrids sterility 4) Tuberose: is an important bulbous 3. Low multiplication rate ornamental plant of tropical and 4. Long juvenile period subtropical areas. Techniques for Overcoming Pre- These are commercially cultivated for loose Fertilisation Barriers flower and cut flower trade and also for the  The cut style method: one day after extraction of its highly valued natural flower stigmatic receptivity, style of flower is cut oil. Dichogamy and Self-incompatibility are 1-2mm above the ovary then pollen occurring. Perianth are funnel shaped, fragrant, applied on cut surface. waxy white, 25 mm long. Stamens = six, ovary  The grafted-style method: Style from = 3 locular, ovules numerous, fruits are pollinated flower after one day attached to capsule. the ovary of desired another flower.  Polianthes geminifera x P. bulliana.  Heat treating of style  Polianthes x blissii- Polianthes  use of plant growth regulators geminiflora x P. tuberosa. Techniques for Overcoming Post-  P. bundrantii = P. tuberosa x P. Fertilisation Barriers: howardii  In vitro rescue method 5) Tulip: - Flower- Hypogyanous, erect,  Embryo culture Perianth (6) segments, stamens (6), ovary  ovary-slice culture superior. Stigma three lobed, Fruit –capsule,  ovule culture Seeds numerous, flat. Tuyl and Creij, 2006 Copyright © March-April, 2019; IJPAB 281

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 reported that by crossing Tulipa gesneriana x production areWedgwood, Ideal, Professor, Tulipa fosteriana -Darwin Hybrid (Triploid). Blue Magic. T. gesneriana and T. kaufmanniana - Partial Role of Mutation in bulbous ornamental resistance crops15.  Tulip Breaking Virus resistance:- Mutation is a sudden heritable change in a Cantata, Princeps characteristic of an organism. Mutations occur  New varieties:- Mughal, Don, Prince in natural populations at a low rate; these are Victoria and Blenda known as spontaneous mutations. Utilization 6) Iris: -is native of Holland, Spain and of induced mutations for crop improvement is England. These are highly suitable for group known as mutation breeding. Mutation have planting, borders and cut flowers. The bulbs certain general characteristics are recessive, are planted in September – October about recurrent, random and deleterious. Flower 10cm deep. Flowering occurs during February shape, plant type, early blooming, flower – March. Important varieties are White colour, long stem, leaf color, neutrality to Excelsior, White Emperor, Sapphire Beauty, photoperiod are the main attributes of the plant and Purple Sensation. Cultivars for year round which are affected by mutation.

1) Gladiolus :- Four mutants3 Cultivar Mutagen Parent Earlier colour Changed colour

Gamma rays 1. Shobha Wild Rose Roseine purple Shell pink (10Gy)

2. Tambari Gamma rays Oscar Single Altered flower colour

3. Swarnima Spontaneous Dhanvantari Light yellow Coppery yellow

Patil12, studied Influence of gamma radiations delayed in spike initiation with decrease in from Co-60 was studied in three varieties of number of florets while lower doses responded gladiolus (Gladiolus hybrida L.) namely, positively. Three desirable mutants with light American Beauty, Nova Lux and Eurovision colours were isolated from all three varieties in irradiated with 1, 2, 3, 4, 5, 6 and 7 kR doses. 5 kR whereas one mutant have bifurcated Percentage of sprouting was affected spike at 6 kR from cv. American Beauty. significantly at1 kR to 4 kR. LD50 was found 2) Dahlia:- to be beyond 7 kR dose for both sprouting. Mutation is a natural or artificially induced Doses of 4 kR and above proved to be change of genetic information contained in a detrimental for various vegetative and floral cell. It occurs when the order of gene is traits. Plants treated with 6 kR and 7 kR did changed or damaged. This can happen not produced flower spikes in cv. Nova Lux sexually in the ovule through the chance and Eurovision whereas cv. American Beauty mating of dissimilar chromosomes. However, had produced few flower spikes. When corms mutants can be induced by irradiation or were treated with higher doses, plant height chemically interference. Juanita provides a was reduced significantly and leaves become good example of natural mutation. Dohare et narrow and leathery. Colour variations in al., located a sectorial chimera in White Pearl florets and whole spike were also increased which has a deep magenta steaked flower and with increase in dose rate along with increase named as Manali. Some Induced mutants in or decrease in number of floral organs. dahlia. Radiation treatments at higher doses caused

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051

Mutants Parent

Bichitra Kenya

Black Beauty Black Out

Happiness Croydon Monarch

Jayaprakash Croydon Apricot Jubilee Kenya Jyoti Kenya Netaji Eagle Stone Pearl Eagle Stone

Dwivedi and Banerji, 2008 studied effect of they recommend 500 and 1000 rads dose of gamma irradiation on dahlia cv “ Pinki” in this gamma rays for induction of somatic mutation. rooted cuttings of dahlia were irradiated with 3) Tuberose: - NBRI (Luck now) 0, 250, 500, 1000 and 1500 rads of gamma releases two mutants i.e. Rajat Rekha rays. Reduction in survival percentage, plant (single)- silvery white streaks along the middle height, and flower size was observed after of the blade. irradiation and with increase in exposure of Swarna Rekha (double)-golden-yellow gamma rays. Morphological abnormalities in streaks are present along the margins of the leaf increased with increase in exposure to blade (1 to 5 kr). gamma rays. On the basis of his observation 4) Tulip18.

Mutants Parents Characters

Faraday (De Mol, 1936) Fantasy White, flushed salmon pink colour

Estella Rijnveld (1954) Red Champion Red flamed, white flower colour

Apeldoorn (Mutants) - Half of one petal being yellow

Bartigon, William Copland, - - Murillo

ICAR in 2013 reported that when they treated embryo into a viable plant, it produce the varieties of Apeldoorn, Golden Melody interspecific and intergeneric hybrids and Strong Gold with different doses of 5, 10 Northern Beauty, Starbrust Sensation is and 20 Gy gamma rays and got results are at developed through embryo rescue in lilium higher dose large number of flower buds dried Asano. before opening, less number of bulbs and  Genetic engineering: - artificial transfer bulblets of gene(s) from one organism to another to Non- Conventional Techniques:- are non- produce novel traits in the recipient living traditional method includes biotechnological organism. tools, tissue culture, genetic engineering, Methods of gene transfer: there are two molecular markers and takes short time, save methods:- labour etc. Direct method: which includes Chemical  Anther culture: - Using anthers to culture Mediated Gene Transfer, Microinjection, haploid plantlets. Discovered by Guha and Electroporation, Particle Gun/Particle, Maheshwari Bombardment Liposome Mediated Gene  Embryo rescue:- is to promote the Transfer Or Lipofection. development of an immature or weak

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Indirect:- Agrobacterium tumefacians, Direct gene transfer:- Agrobacterium rhizogenes

Methods Description

Chemical mediated gene Polyethylene glycol (PEG) and Dextran Sulphate induce DNA uptake into transfer plant protoplasts.Calcium phosphate

Microinjection DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) fine tipped (0.5 - 1.0 micrometer diameter) glass needle or micropipette.

Electroporation High voltage electrical pulses to a solution containing a mixture of protoplasts and foreign DNA introduce DNA which enters the cells through reversible pores created in the membrane by the action of short electrical pulses.

Particle gun Foreign DNA containing the genes to be transferred is coated onto the surface (microprojectile gun) of minute gold or tungsten particles (1-3 micrometers) Biolistics inventor Sanford (1988)

Liposome mediated gene Circular lipid molecules with an aqueous interior that can carry nucleic acids. transfer or lipofection Encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. DNA enters the cell and then to the nucleus

In direct gene transfer:- from plants that had been grown in the dark Agro bacterium-mediated gene transfer : is for at least 2 months. a soil bacterium helps in transferring a piece of Otani et al.10, studied on establishment DNA (T-DNA) into the genome of host plants. of Agrobacterium-mediated genetic Genes inserted into the T-DNA region through transformation system in Dahlia. They use Ti plasmids are co-transferred and integrated mass of shoot primordia (MSP) induced on into the host genome. MS medium. The test was Confirmation PCR Ozel and Kamo11. studied on and Southern blot analyses. Agrobacterium Agrobacterium-mediated transformation of tumefaciens strain EHA101 (pIG121-Hm) Easter Lily (Lilium longiflorum „Nellie harboring both β-glucuronidase (GUS) and White‟). Plants were grown In vitro on hygromycin resistant genes.Shoots were Murashige and Skoog‟s medium they use A. successfully regenerated from hormone-free tumefaciens strain AGL1 containing medium without hygromycin and they rooted pCAMBIA2301 uidA gene that codes for β- on hormone-free medium containing glucuronidase was cultured with kanamycin. hygromycin. Hygromycin-resistant plants thus Precultured explant was exposed to obtained showed histochemical blue staining Agrobacterium incoluation for 24-25 mins for GUS. after etoilted for 11 days at 250C. extra Dilip et al.5, studied on Genetic agrobacterium rinsed from the explant and transformation of Fusarium oxysporum f sp. then cultured on co-cultivation medium gladioli with Agrobacterium. They use containing 500 mg/L cefotaxime. Results are Agrobacterium tumefaciens containing using smaller bulb scales from the inner region of the AGL-1 strain Confirmation test by PCR the bulb or from a small bulb showed and Southern blot analyses. Vectors are significantly more blue spots than the larger hygromycin B phosphotransferase (hph) gene bulb scales. Maximum GUS expression and fluorescence reporter genes EGFP (green), occurred when bulb scales had been obtained EYFP (yellow), ECFP (cyan) Result are

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Gladiolus successfully generated several stable developed in this study will be useful in transformed lines showing hygromycin B molecular and histopathological investigations. resistance and green, yellow or cyano Morphological traits as a target for genetic fluorescence.The transformed Fog lines modification

Crop Gene Trait Reference

Caladium Maize Lc+C1 Leaf color Li et al, 2005

Lilium Rol A,B,C Dwarf Mercuri et al, 2003

Lilium Maize Zm401 Pollen-less Li et al, 2005

Lilium Carotenoids Flower, leaf color Azadi et al., 2010

Narcissus Phytoene synthase Flower color Lu et al., 2007 Virus resistant Genes:-

Type of gene Resistant Gene Virus

Coat protein gene CP-TMV Tobacco mosaic virus

CP-AMV&TRV α –α mosaic virus & Tobacco rattle virus

CP-CTV Citrus tristeza virus

CP-PPV Plum pox virus

Nucleoplasmid Gene NC-TSWV Tomato spotted wilt virus

Azadi et al.2, studied on increased resistance to  Expands useful genetic diversity for crop Cucumber Mosaic Virus (CMV) in Lilium improvement transformed with a defective CMV replicase  Increases favorable gene action gene. Cultivar: Acapulco. They use  Independent of environmental factors Agrobacterium tumefaciens mediated (CMV2- 1. Morphological markers: Traditionally, GDD) gene. They Confirm test by reverse diversity within and between populations was transcription PCR (RT-PCR). Result are determined by assessing differences in increased levels of resistance to CMV was morphology. These measures have the observed in Lilium, and CMV-GDD Replicase advantage of being readily available, do not gene is an effective construct that has require sophisticated equipment and are the protection against CMV in Lilium. most direct measure of phenotype, thus they Marker is the sign and trait to identify any are available for immediate use, an important individual. There are three types of markers4. attribute. However, morphological Morphological markers, Protein (Biochemical) determinations need to be taken by an expert markers and DNA (molecular) markers. in the species, they are subject to changes due NEED OF MOLECULAR TECHNOLOGY to environmental factors and may vary at  Save time and other resources: save time different developmental stages and their compared to phenotypic selection (PS), number is limited. E.g Albinism, Plant more popularly known as conventional height, leaf morphology. breeding. In PS, breeders must wait until 2. Protein (biochemical) markers: To the plant is fully mature so they can overcome the limitations of morphological identify desired traits by observing the traits, other markers have been developed at plant. both the protein level (phenotype) and the  Eliminate unwanted plants DNA level (genotype). Protein markers are Copyright © March-April, 2019; IJPAB 285

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 usually named biochemical markers but, more combined genotype of the two and more; they are mistakenly considered as a homozygous parents. common class under the so-called „molecular  Evenly distributed throughout the markers‟. genome. The more distributed and dense Protein markers (seed storage proteins genome coverage is, the better the and isozymes) are generated through assessment of polymorphism. electrophoresis, taking advantage of the  Discriminating, that is, able to detect migrational properties of proteins and differences between closely related enzymes, and revealed by histochemical stains individuals. specific to the enzymes being assayed.  Not subject to environmental influences. Detecting polymorphisms i.e. detectable The inference of a marker‟s genotype differences at a given marker occurring among should be independent of the environment individuals in protein markers is a technique in which the individual lives or its that shares some of the advantages of using developmental stage. morphological ones. However, protein markers  Neutral. The allele present at the marker are also limited by being influenced by the locus is independent of, and has no effect environment and changes in different on, the selection pressure exerted on the developmental stages. Even so, isozymes are a individual. This is usually an assumption, robust complement to the simple because no data are usually available to morphometric analysis of variation. confirm or deny this property. 3. DNA (molecular) markers  Inexpensive, Easy, fast and cheap in DNA polymorphisms can be detected in detecting across numerous individuals. If nuclear and organelle DNA, which is found in possible, the equipment should be of mitochondria and chloroplasts. Molecular multipurpose use in the experiment. markers concern the DNA molecule itself and, Molecular markers: as such, are considered to be objective Strauss, et al.17, distinguished between two measures of variation. They are not subject to classes of molecular marker viz. molecular environmental influences; tests can be carried genetic markers (those derived from direct out at any time during plant development; and, analysis of polymorphism in DNA sequences), best of all, they have the potential of existing and biochemical markers (those derived from in unlimited numbers, covering the entire study of the chemical products of gene genome. expression). Characteristics of good marker:  Molecular markers consist of specific  Polymorphic, that is, it is variable among molecules, which show easily detectable individuals. The degree of polymorphism differences among different strains of a detected depends on the technology used species or among different species. to measure it.  Molecular markers analyze genes directly.  Reproducible in any laboratory  Molecular markers reveal neutral sites of experiment, whether within experimental variation at the DNA sequence level. events in the same laboratory or between These variations do not necessarily show different laboratories performing identical themselves in the phenotypes. experiments.  Use of linked molecular markers would  Co dominant. Depending on the type of allow indirect selection for desirable traits application, the selected technology must in early segregating generations at the be able to detect the marker‟s different seedling stage and independent of forms, distinguishing between environmental influences. homozygotes and heterozygotes  This, in turns will save time and other (codominant inheritance). A heterozygous resources that are needed. individual shows simultaneously the Copyright © March-April, 2019; IJPAB 286

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051  They have the big advantage that they are considered the most widely used molecular much more numerous than morphological marker type in molecular studies (White et al markers. 07). It is a first PCR-based marker. The Classification of molecular markers based random amplified polymorphic DNA (RAPD) on the basic strategy technique is a PCR-based method that uses a  Non PCR based approaches short primer (usually 10 bases) to amplify – RFLP (Restriction fragment length anonymous stretches of DNA. With this polymorphism) technique, there is no specific target DNA, so  PCR-based approaches each particular primer will adhere to the – RAPD (Random Amplified Polymorphic template DNA randomly. As a result, the DNA) nature of the obtained products will be – AFLP (Amplified fragment length unknown. The DNA fragments generated are polymorphism) then separated and detected by gel – SSR (Simple Sequence Repeat) electrophoresis. RAPDs can be detected by – ISSR ( Inter-simple sequence repeats) running PCR products through electrophoresis – SCAR (Sequence characterized amplified on an agarose or acrylamide gel. regions) In both cases, the gel is stained with ethidium – CAPS (Cleaved amplified polymorphic bromide. The difference obtained by running sequence ) RAPD products in acrylamide versus agarose RFLP (Restriction fragment length lies only in the degree of resolution of bands. polymorphism) (Non PCR) In most cases, agarose gel electrophoresis  Restriction fragment length polymorphism gives sufficient resolution. (RFLP) analysis was one of the first Main features: techniques to be widely used for detecting Specific regions of the DNA molecule are variation at the DNA sequence level. The amplified for analysis of variation by the PCR, principle behind the technology rests on using primers (usually 10 bases), the possibility of comparing band profiles  Random stretches of the genome will be generated after restriction enzyme sampled and amplified and used as the digestion in DNA molecules of different basis for variation analysis individuals. Diverse mutations that might  The no. of amplified fragments generated have occurred affect DNA molecules in by PCR will depend on the length and different ways, producing fragments of sequence of the primer and the genome variable lengths. These differences in size fragment lengths can be seen after gel  In most plant s a 10 nucleotide primer will electrophoresis, hybridization16 and generate on an average 2-10 amplification visualization. products20. Procedure Many different fragments (corresponding to  Genomic DNA digested with Restriction multiple loci dispersed throughout the Enzymes. genome) are normally amplified, using each  DNA fragments separated via single primer. The technique is therefore rapid electrophoresis and transfer to in detecting polymorphisms. Although most nitrocellulose membrane. commercially produced primers result in  Membranes exposed to probes labelled several fragments, some primers may fail to with P32 via southern hybridization. give amplification fragments from some Film exposed to X-Ray. material. The technique is simple. RAPD Random Amplified Polymorphic DNA analysis does not require expertise to handle (RAPD) technique hybridization of DNA or other highly technical First developed independently by Welsh & activities. McClelland21 and Williams et al., and Copyright © March-April, 2019; IJPAB 287

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Amplified fragment length polymorphism or simple sequence repeat polymorphisms (AFLP) (SSRPs). The term “Microsatellites” was This is a highly sensitive method for detecting coined by Litt and Lutty. SSRs are short polymorphism throughout the genome and it is tandem repeats, their length being 1 to 10 bp, becoming increasingly popular. It is essentially most typically, 2-3 bp. SSRs are highly a combination of RFLP and RAPD methods, variable and evenly distributed throughout the and it is applicable universally and is highly genome. This type of repeated DNA is reproducible. It is based on PCR amplification common in eukaryotes, their number of of restriction fragments generated by specific repeated units varying widely among restriction enzymes and oligonucleotide organisms to as high as 50 copies of the adapters of few nucleotide bases19. it is a repeated unit. These polymorphisms are novel DNA fingerprinting technique. DNA identified by constructing PCR primers for the fingerprinting involves the display of a set of DNA flanking the microsatellite region. The DNA sample. Fingerprints are produced flanking regions tend to be conserved within without prior sequence knowledge, using a the species, although sometimes they may also limited set of genetic primers. AFLP technique be conserved in higher taxonomic levels. uses stringent reaction conditions for primer PCR product size variation is caused by annealing and combines the reliability of differences in the number of microsatellite RFLP technique with the power of PCR repeat units. SSR polymorphisms can be technique. visualised by agarose or polyacrylamide gel Interpreting AFLP bands: electrophoresis. Microsatellite alleles can be  The AFLP technique detects detected, using various methods: ethidium polymorphisms arising from changes bromide, silver staining, radioisotopes or (presence or size) in the restriction sites or fluorescence. adjacent to those. If fluorescence-labelled primers are used, and  Different restriction enzymes can be used, the products are different enough in size and and different combinations of pre- and not overlapping, then multiplexing that is, selective nucleotides will increase the loading more than one sample per lane of probability of finding useful reaction products can greatly increase the polymorphisms. already high efficiency of these markers.  The more selective bases, the less Inter-simple sequence repeats (ISSRS) polymorphism will be detected.  Reported by Zietkiewicz et al27.  Bands are usually scored as either present  They are regions found between or absent. microsatellite repeats.  Heterozygous versus homozygous bands  The technique is based on PCR may be detected, based on the thickness of amplification of inter-microsatellite the signal, although this can be tricky. sequences. Procedure  Because of the known abundance of repeat DNA is digested with two different restriction sequences spread all over the genome, it enzymes. Oligonucleotide adapters are ligated targets multiple loci. to the ends of the DNA fragments. Specific Application of molecular marker in subsets of DNA digestion products are floriculture: amplified, using combinations of selective  Genetic diversity analysis primers. Polymorphism detection is possible  Disease diagnostics with radioisotopes.  Gene mapping Microsatellites (SSRs, STMS or SSRPs)  Marker assisted selection Microsatellites are also called simple  Marker assisted backcrossing sequence repeats (SSRs) and, occasionally,  Identification of qtls / marker assisted sequence-tagged microsatellite sites (STMS) pyramiding Copyright © March-April, 2019; IJPAB 288

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 Institutes working on molecular markers in floricultural crop IIHR Genetic diversity analysis in Bougainvillea and Carnation

Molecular markers for Bacterial blight resistance in Anthurium andreanum

NBRI Molecular characterization of plant viruses affecting ornamental plants.

Varietal identification and assessment of genetic relationships in bulbous plants

IARI Genetic diversity analysis in Gladiolus cultivars (NBPGR)

IHBT Diversity analysis and characterization of viruses affecting ornamentals (Palampur) Use of Molecular Markers in Bulbous plants

Crop Marker used References

Gladiolus Morphological and RAPD markers Pragya et al, 2010 (Genetic diversity)

Lilium RAPD Benedetti et al, 2000

Gladiolus RAPD (Fusarium Wilt) Nasir et al, 2012

Alstroemeria AFLP (Genetic diversity) Han et al, 2000

Tulip SNP Markers (Genetic mapping of Tang et al, 2015 resistance to Fusarium oxysporum )

Tuberose Morphological and ISSR markers Kameswari et al, 2014 (Genetic diversity )

Marker assisted selection (MAS):- cultivar Friendship (CV-1) was extracted It is the selection of trees with desirable traits using the CTAB method 29 RAPD primers based on their molecular genotype. It can be were used for screening.The amplified used alone or in combination with the classical products varied from 200bp to 1800bp. 5 methods of selection. primers were found to be polymorphic and Benefits: produced different percentages of  Early selection that can potentially polymorphism. The average number of decrease the breeding cycle time. fragments per primer was 6 from which 62%  Decrease cost by reducing expensive were found to be polymorphic fragments progeny test establishment, maintenance Molecular assisted breeding for disease and measurement.Increasing selection resistance in lily7. intensity because more individuals can The variation in resistance to Fusarium was possibly be evaluated. determined in four different green house tests  Increasing the relative efficiency of in four different years on the same 100 selection on low heritability traits descendants of a backcross population. Asiatic Molecular analyses of Gladiolus lines with hybrid lilies 'Connecticut King' and 'Pirate' improved resistance against Fusarium wilt8. were crossed. One F1 hybrid, the cultivar Procedure:- 'Orlito', was used as father in a backcross with DNA from leaves of In-vitro selected, Gamma 'Connecticut King„.„Connecticut King' is irradiated cell lines and control of Gladiolus partially resistant to Fusarium wilt.15 AFLP

Dogra and Dhatt Int. J. Pure App. Biosci. 7 (2): 279-291 (2019) ISSN: 2320 – 7051 primers were used for analysis in 527 varieties. segregating population.Of the 527 segregating  In India also similar sort of coordinated markers in the 'Connecticut King' x 'Orlito' efforts are required to test the suitability cross 176 were originating from 'Connecticut and application of these marker techniques King„201 from 'Orlito and remaining 150 for molecular profiling of varieties and markers were present in both parents and parent lines of hybrids. segregated in the offspring population.  Molecular marker technologies can be BEST DNA MARKERS FOR VARIOUS used to attack trade secrets by rapid APPLICATIONS: identification of female parent inbred line Applications that require large number of contaminants in bags of hybrid seed. loci: These inbred lines might then be used  Measuring genetic diversity and directly as parents of hybrids or as parents differentiation. for further breeding.  Estimating rates of gene flow or migration  Molecular marker technology can be used (between population). to identify segregating molecular  Genetic linkage mapping or quantitative characteristics in an otherwise uniform trait loci localization. variety and thus to select a distinct “new” Markers of Choice- AFLP’s OR RFLP’s variety from the segregating source Applications that require high without any breeding effort being discrimination power: expended.  Characterizing mating systems.  Microsatellite markers have been used  Analyzing paternity or parentage. successfully to determine the degree of  Characterizing patterns of gene flow or relatedness among individuals or groups of migrating within populations. accessions, to clarify the genetic structure,  Assessing seed orchard efficiency. or partitioning of variation among  Quality control in breeding, DNA individuals, accessions, population and fingerprinting or cross verification. species of rice and similar trends can be Markers of Choice- Microsatellites applied in the field of forestry for the Applications that require DNA sequence registration of hybrids. information:  Phylogeny and taxonomy CONCLUSIONS Markers of Choice- PCR and sequencing  No genetic barriers FUTURE PERSPECTIVES:  Novelty through genetic engineering  DNA fingerprinting, through its high  Speed of improvement precision in identifying plant genotypes,  Altered plant byproduct, form and colour holds considerable promise as a reliable  Creation of genetic variation tool of intellectual property protection of  Easy acceptability than GM food crops crop varieties and germplasm. In order to  Molecular markers provide valuable fully harness the potential of this information about genetic diverstity and technology, it is necessary to establish a used to broaden the gene pool of network of DNA fingerprinting ornamental crops. laboratories using uniform set of standardized protocols for each crop. REFERENCES  protection of crop varieties or for verifying 1. Arora, J. S., Introductory Ornamental varietal identity. It has included Horticulture. Kalyani Publishers: electrophoresis of seed proteins in barley Ludhiana. 170pp. (2010). and wheat and of isozymes in maize, 2. Azadi, P., Otang, N. V., Supaporn, H., soybean and sunflower as additional Khan, R. S., Chin, D. P., Nakamura, I., characters for establishing distinctness of Mii, M., Increased resistance to cucumber Copyright © March-April, 2019; IJPAB 290

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