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Application of Biotechnology forImprovement of ornamentalsBy: Amany Abozaid .

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Ornamental floriculture is becoming an importantindustry .Ornamentals include a large variety of crop plantsCut flowers,Bulbs and corms,Foliage and Flowering pot plants.All the present day ornamental varieties and novelties are as a result of extensive hybridization, induced mutation and selection .

Introduction

Biotechnology :Bio = Biology Technology = Methodology The application of Biology(for the benefit of humans)

Genetic engineering:The technology of preparing recombinant DNA in vitro by cutting up DNA molecules and splicing together fragments from more than one organism.Genetic engineering is a laboratory technique for gene manipulation.Genetic engineering brings about novel combination ofgenes by using recombinent DNA technology which is not possible through natural means.Biotechnology

Genetic engineering of plants is much easier than animals.there is natural transformation system for plants(Agrobacterium).plant tissue can redifferentiate.plant transformation and regeneration are relatively easy for a variety of plants.Agrobacterium tumefaciens can infect wounded plant tissue, transferring a large plasmid, the Tiplasmid, to the plant cell.Genetic engineering

Important methods in recombinant DNA technology are Isolation of desired gene Insertion of isolated gene into a suitable vector Introduction of recombinant vector in to host Selection of transformed host cells (A.C.Dutta 2005)

Genetic engineering

Digestion of the cell wall by enzymatic action, dissolution ofthe biological membranes by detergent losses, centrifugation to isolate pure DNA.DNA cut into no. of fragments by restriction endonulcleases molecular scissors with sticky ends. Genetic engineering contd.Isolation of desired gene:

Basic steps involved in process

Most widely used More economical More efficientAgrobacterium mediated gene transfer Particle bombardment or micro projectile . Direct DNA delivery by PEG . Electroporation . Microinjection .Chandler and Brugliera, 2011Gene transfer methodsDirectIndirect

Electroporation :

1-2 m of tungsten or gold particles (microprojectiles)coated with DNA to be used for transformation are accelerated to velocities using pressurized Helium gas

Particle gun method : ( Klein et al ., 1988 )

Microinjection:

DNA solution is injected directly inside the cell using capillary glass micropipetts .

Production of engineered long-life and male sterile Pelargonium plantsGarca-Sogo et al. BMC Plant Biology 2012, 12:156http://www.biomedcentral.com/1471-2229/12/156

Classification :

Kingdom: Plantae Subkingdom: Tracheobionta Superdivision: Spermatophyta Division: Magnoliophyta Class: Magnoliopsida Subclass: Rosids Order: Geraniales Family: Geraniaceae Genus: Pelargonium

Pelargonium peltatum cv. AranjuezPelargonium zonale cv. 370

Establishment of in vitro cultures

Plant materialSurface sterilizationMorphogenesis Induction Medium (MIM)Elongation Medium RootingAcclimatization

Surface sterilizationMorphogenesis Induction Medium (MIM)Calculate activity cost drivers ratesRootingAcclimatization

Plant materialPelargonium zonale cv. 370 Explants: Young leaf explants from 3040 days old plantlets Pelargonium peltatum cv. AranjuezEstablishment of in vitro cultures

Establishment of in vitro cultures

Plant materialMorphogenesis Induction Medium (MIM)Elongation Medium RootingAcclimatization

Rinsed three times with sterile distilled water. Iimmersion in a 2.5% solution of sodium hypochlorite for 20 min.

Surface sterilization

Establishment of in vitro cultures

Plant materialSurface sterilizationCalculate activity cost drivers ratesRootingAcclimatization

leaves was cut into 1 cm2 pieces and cultured on MIM MS basal medium and Shahin [46] vitamins

Morphogenesis Induction Medium (MIM)supplemented with50 mg l-1 kanamycin

Regeneration in Pelargonium zonale was carried out via direct organogenesis andin Pelargonium peltatum via somatic embryogenesis.

Establishment of in vitro cultures

Plant materialSurface sterilizationMorphogenesis Induction Medium (MIM)RootingAcclimatization

After 2.5 - 3 months in culture, calli showing well developed morphogenetic structures (shoots in the case of P. zonale and somatic embryos in P. peltatum) were transferred to a selective Elongation Medium . Elongation Medium (EM: MS basal medium and Shahin vitamins, supplemented with 50 mg l-1 kanamycin) All explants were subculture every 2 weeks onto the same fresh medium until shoots were long enough to be separated ..

Elongation Medium

Establishment of in vitro cultures

Plant materialSurface sterilizationAssign costs to activity cost poolsElongation Medium Acclimatization

After 1 1.5 months in EM, the shoots were cut and cultivated in Rooting Medium (RM).

Rooting

Establishment of in vitro cultures

Plant materialSurface sterilizationMorphogenesis Induction Medium (MIM)Elongation Medium Rooting

and acclimatized in growth chambers under (16-h light/8-h dark photoperiod) and then transferred to a greenhouse until they flowered.. Regenerated plantlets with welldeveloped roots were transferred to plastic pots containing peat moss and perlite (3:1).

Acclimatization

Transformation efficiency was estimatedas the number of independent transformationevents (one transgenic plant per explant) in relation to the total number of inoculated explants.

Cytokinins have been implicated in several aspects ofplant development, including plant senescence [15-20],and are thought to be synthesized mainly in the rootsand transported to the shoots via the xylem.

Overexpression of the ipt gene in transgenic plants led to elevated foliar cytokinin concentrations and delayed leaf senescence, but high cytokinin levels have been reported to be detrimental to growth and fertility [26 30].To circumvent these effects : Specificgene promoter (pSAG12 )Isopentenyl phosphotransferase (IPT)

Induced GMS

Promoter which induces transcription in male reproductive specifically

Gene which disrupts normal function of cell

Agrobacterium-mediated transformation

regeneration

male-sterile plant

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(A portmanteau of "BActerial" "RiboNucleASE")

is a bacterial protein that consists of 110 amino acids and has ribonuclease activity. It is synthesized and secreted by the bacterium

Bacillus amyloliquefaciens, but is lethal to the cell when expressed without its inhibitor barstar .

The inhibitor binds to and occludes the ribonuclease

active site, preventing barnase from damaging the cell's RNA Barnase

LBA4404 cells were electroporated to carry different plasmids a pBIN19 binary vector . A. tumefaciens strain and chimaeric gene constructs

T- DNA region

Bacterial DNA

Virulence region

Oregion of replicationPBiN19

Organogenesis - Pelargonium zonale

Somatic embryogenesisin Pelargonium peltatum

Transformed explants were examined periodically for gfp expression under a fluorescence stereomicroscope (Leica MZ FLIII) .

Light microscopy

PCR analysis

Plant DNA used for PCR analysis was extracted from young leaves using the protocol of Rogers and Bendich [50] .PCR products were detected by UV light after electrophoresis on 1% w/v agarose ethidium bromide gels.

Identification of the ipt transgene (460 bp fragment) by PCR in different P. zonale pSAG12::ipt transgenic plants. C + (positive control: pVDH393-pSAG12::ipt) and TI (negative control).

Identification of the barnase-barstar transgene (544 bp fragment) by PCR in different P.zonale male sterile plants. C + (positive control: pBI101-PsEND1::barnase-barstar) and TI (negative control).

Realtime RT-PCR analysis of pSAG12::ipt transcript levels in detached leaves from the transgenic lines 3.4, 3.9, 4.3 and 4.12. Each samples expression level relative to Pelargonium x hortorum PhACTIN7 is the mean of three biological repeats. C: control WT leaves.

Measurements were taken in the greenhouse on transgenic plants and WT control plants :

Plant height (distance from soil line to top of the tallest growing point), leaf length and width (average measurements from five fully expanded leaves),leaf petiole length, internodal length Number of inflorescences per plant were evaluated.

Morphological measurements were taken over the course of several days on each plant as its first five flowers reached anthesis . Means differing significantly were compared at a 5% probability level. Data variability was expressed as the mean SE.Morphological measurements

inflorescence reversion

(a), 6 (b), 8 (c), 17 (d), 22 (e), 24 (f), 27 (g) and 34 (h) days of incubation in darkness.

Analysis of leaf senescence was conducted by extraction of chlorophyll in detached leaves incubated in darkness from WT control and pSAG12::ipt plants respectively.Using a porcelain mortar cooled with liquid nitrogen,samples were crushed to a fine powder. In 10 ml centrifuge tubes the samples were mixed with 100 mg of MgCO3 and 5 ml of 100% (v/v) acetone. Bleached leaf material was removed by centrifugation (5 min; 2,000 g) and 1 ml aliquots of supernatants transferred to new tubes. Chlorophyll (a + b) content of extracts was determined spectrophotometrically [53].Quantification of chlorophyll

(i) Mean concentration (SE) of chlorophyll a + b (mg/g fresh weight) from detached leaves of control (WT) and pSAG12::ipt (TRG) plants at 0, 6 and 8 days of incubationin darkness .

(j) Senescence delay of detached leaves from pSAG12::ipt plants. Fresh weight changes in detached leaves of WT P. zonale and a transgenic line carrying the pSAG12::ipt chimaeric gene over the time course analyzed. Data are the means of sixteen leaves SE. Bars: 1 cm.

Conclusions

The chimaeric pSAG12::ipt construct useful in Pelargonium spp. to delay the senescence process and to produce long-lived plants, which could have commercial interest.Transgenic pSAG12::ipt plants showed delayed leaf senescence, increased branching and reduced internodal length as compared to non-transformed plants.Transgenic pSAG12::ipt plants showed a more compact architecture than the WT.

Conclusions

Expression of the barnase gene under control of PsEND1 promoter caused specific ablation of the tissues, necrotic at early stages of anther development. No pollen grains were observed in the ablated anthers from the male-sterile plants, indicating that barnase effectively destroys specific cell lines that form the structural tissues of the anther , preventing pollen development. .

The use of engineered male sterility would be especially useful to eliminate pollen allergens and to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species.

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References

Thank YouAmany Abozaid

Thank You(Basic)

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