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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