Biol328 - B3212 Molecular Biotechnology...Lecture 18 •5/29/2018 •2 ... • Half-life varies from...
Transcript of Biol328 - B3212 Molecular Biotechnology...Lecture 18 •5/29/2018 •2 ... • Half-life varies from...
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Prof. Fahd M. Nasr
Faculty of SciencesLebanese University
Beirut, Lebanon
https://yeastwonderfulworld.wordpress.com/
Biol328 - B3212Molecular
BiotechnologyLecture 18
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Plant Biotechnology
RNA issues• Similar to other eukaryotes• End modifications stability +
translation• Half-life varies from minutes days• Destabilisation sq DST in the 3'UTR
– SAUR transcripts induced by the hormone auxin are short-lived mRNAs
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Implications for plant transformation
• Stable integration of transgenes• Appropriate expression
– Spatially and temporally– Proper processing of transcripts and proteins
• Strong promoter for constitutive expression (CaMV 35S promoter)– Timing and location are not critical
• Inducible or controlled expression
Promoters used for expression in transgenic plants
Cauliflower mosaic virus 35S promoter
CaMV 35S is a strong promoter that is active in essentially all dicot plant tissues
CaMV is a circular dsDNA genome virus
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Genomic map of CaMV
Arabidopsis: a model plant• Small dicotyledonous cruciferous plant• A weed with no commercial value• Mustard family related to cabbage, canola,
cauliflower, …• Short life cycle, small stature, large
numbers of offspring• Suited for genetics (mutational analysis)• Small genome 125Mb
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Arabidopsis thaliana• Small flowering plant a model organism in plant biology• Member of the mustard (Brassicaceae) family• No major agronomic significance• Offers important advantages for basic research in genetics and
molecular biology– ~ 115 Mb of the 125 Mb genome has been sequenced and annotated– Extensive genetic and physical maps of all 5 chromosomes– The life cycle is short--about 6 weeks from germination to seed maturation– Plant easily cultivated in restricted space prolific seed production– Transformation is efficient utilizing Agrobacterium tumefaciens– A large number of mutant lines and genomic resources is available– Studied by a multinational research community in academia, government
and industry
Arabidopsis thaliana
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Arabidopsis thaliana
Arabidopsis genome (AGI) • 125Mb in 5 chromosomes• ~25,000 coding genes• Gene density (kbp per gene) is ~4.5• >70% have homologs in other species• 350-400 rRNA gene units (2 and 4)• 589 tRNA genes• Mt genome 367kbp, 58 genes• Chloroplast genome 154kbp, 79 genes
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Genomics• A discipline in genetics study of the genomes of
organisms– Fine-scale genetic mapping– Determine the entire DNA sequence– Intragenomic phenomena gene interactions– A broader scope of scientific inquiry associated
technologies– The study of all the genes DNA (genotype), mRNA
(transcriptome), or protein (proteome) levels
Omics disciplines• Refers to a field of study in biology ending
in -omics– Genomics, proteomics, metabolomics, etc.– The suffix -ome to a totality of some sortGenome, proteome, metabolome, etc.
– Functional genomics functions of as many genes as possible of a given organism combines different -omics techniques transcriptomics and proteomics
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Kinds of omics studies• Transcriptomics transcriptome: mRNA, rRNA, tRNA,
and other non-coding RNA• Proteomics proteome + modifications large-scale
study of proteins structures and functions• Functional genomics gene and protein functions and
interactions• Metabolomics chemical processes involving metabolites small-molecule metabolite profiles
• Structural Genomics 3-dimensional structure of every protein experimental and modeling approaches
Kinds of omics studies• Immunoproteomics proteins involved in the immune
response• Epigenomics epigenome complete set of epigenetic
modifications• Metagenomics Study of metagenomes genetic
material recovered directly from environmental samples• Nutrigenomics effects of foods and food constituents on
gene expression• Toxicogenomics information about gene and protein
activity in response to toxic substances
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Kinds of omics• Cognitive genomics, Comparative genomics,
Epigenomics, Functional genomics, Genomics, Immunoproteomics, Metabolomics, Metabonomics, Metagenomics, Nutrigenetics, Nutrigenomics, Nutriproteomics, Nutritional genomics, Personal genomics, Pharmacogenomics, Pharmacomicrobiomics, Proteogenomics, Proteomics, Psychogenomics, Stem cell genomics, Structural Genomics, Toxicogenomics, Transcriptomics, etc.
Biotechnological implications• AGI is a milestone in plant biology• Arabidopis thaliana is not a crop plant• Processes share common features in A.
thaliana and crop plants– Stress tolerance, pest resistance, …
• A. thaliana: model to study these processes• Rice genome (and other crops)
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Plant tissue culture• Methods of plant transformation
– Regeneration of the whole plant (GM crop) from isolated plant cells
– In vitro regeneration (optimal conditions)– Plant cells are accessible to gene transfer– High freq of regeneration does not
correlate with high freq of transformation
Plasticity and totipotency• Plasticity
– Plants endure extreme conditions– Processes (growth and development) adapt to
the environment– Initiate cell division from any plant tissue
• Totipotency– Maintenance of genetic potential– All plant cells (correct stimuli) express the total
genetic potential
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Plant cell culture media• Essential elements (mineral ions)
– Macroelements (large amounts)– Microelements (trace amounts)– Iron source (iron sulphate)
• Organic supplement (vitamins and a.a.)
• Source of fixed carbon (sucrose)
Plant growth regulators• Are critical components
developmental pathways of plant cells• Plant hormones
– Auxins– Cytokinins– Gibberellins– Abscisic acid– Ethylene
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• Auxins– Promote cell division and growth– IAA (indole-3-acetic acid) is naturally occurring
but unstable– Chemical analogues, more common, stables
• Cytokinins– Promote cell division– Zeatin and 2iP (2-isopentyl adenine), expensive
and unstable– Synthetic analogues are used
Plant growth regulators
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• Gibberellins– Regulation of cell elongation– Determine plant height and fruit-set– Naturally occurring, few are used
• Abscisic acid– Inhibits cell division– Promote somatic embryogenesis
• Ethylene– Controls fruit ripening
Plant growth regulators
Plant hormones
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• Difficulty in predicting their effects• Culture response varies between species and
cultivars• Some principles paradigm
– Auxins and cytokinins are used together– Ratios determine the type of regeneration– High A to C favors root formation– High C to A favors shoot formation– A=C favors callus production
Plant growth regulators and tissue culture
Culture types• Initiated from sterile pieces (explants)• Explants
– Pieces of organs (leaves or roots)– Specific cell types (pollen, endosperm)– Features: younger, rapid growth, ..
• Different types– Callus– Cell-suspension cultures– Protoplasts and others …
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CallusMajor Plant Hormones
Auxins Cytokinins Structurally related to adenine
auxin < CYTOKININ shootsAUXIN > cytokinin roots
auxin = cytokinin undifferentiated callus
Produced by actively growing tissuesparticularly roots, embryos, and fruits
Natural auxin is indoleacetic acid (IAA)
Apical meristem is the major site of auxin synthesis
Callus Nicotiana tabacum
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Produce callus transform callus stimulate shooting by cytokinin addition
+ cytokinin
Callus• Unorganized, growing and dividing mass of
cells (A = C)• Any plant tissue explant• Continuous proliferation subculture• Dedifferentiation morphology and
metabolism• Cells lose their ability to photosynthesize• Performed in the dark
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• Habituation process culture loses the requirement for A and/or C
• Manipulation of the A to C ratios development of shoots, roots or somatic embryos regeneration of the whole plant
• Can be initiated from cell suspensions
Callus
• Callus cultures fall into two categories– Compact callus
Cells are densely aggregated– Friable callus
Cells are loosely associatedCallus is soft and breaks apart easilyProvides the inoculum cell-suspension
cultures
Callus
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• Keep the culture fresh repeat subculturing
• After subculture cells divide biomass increases– Nutrients are exhausted– Build up of toxic by-products– Entry into stationary phase (cells die)– Cells are transferred before entry into SP
Cell-suspensions cultures (CSC)
Protoplasts• Plant cells without cell wall• Isolated from leaf mesophyll or cell
suspensions• Two approaches
– Mechanical low yield, poor quality, ..– EnzymaticCell wall degrading enzymes (cellulase+pectinase)Hypertonic solution
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Protoplasts of cells from a petunia's leaf
• Fragile and easily damaged• Shallow liquid medium aeration• Plated onto solid medium callus• Whole plants regenerated by
organogenesis or somatic embryogenesis
• Can be transformed by a variety of means
Protoplasts
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• Root cultures– Explants from the root tip
• Shoot tip and meristem culture– Shoot apical meristem (clonal propagation)
• Embryo culture– Embryos as explants callus cultures or
somatic embryogenesis• Microspore culture
– Pollen or anthers as explants (haploid)
Other culture types
Plant regeneration
• How the whole plant can be regenerated?–Somatic embryogenesis
Similar to zygotic embryo germination
–Organogenesis
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Somatic embryogenesis• Asexual embryogenesis• Embryo-like structures (from somatic
tissues) plants• Direct and indirect somatic embryos
– Direct: embryo cell or small group of cells
– Indirect: embryo callus tissue or cell suspension callus explant
Single cell
Group of cells
Globular embryo
Heart-stage embryo
Torpedo-stage embryo
cotyledonsSAM
RAM
SomaticEmbryogenesis
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Soybean somatic embryos
Somatic embryogenesis from grape callus
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Organogenesis• Production of organs
– Explants or callus culture• Relies on the inherent plasticity of
plant tissues
The end