Plant Tissue Culture

Laboratory Exercise – Seed germination of Bean and Corn

What is asexual plant propagation?

• Identical offspring are produced by a single parent

• Continuous sexual reproduction – risk producing plants that have lost desirable traits and gained unwanted ones

Introduction to Plant Propagation

• Asexual reproduction by Cuttings

• Cuttings are pieces of stems, leaves or roots placed in appropriate media – vermiculite, sand, perlite, potting soil or water

• Existing cells in growing tissue will develop into missing roots, stems or leaves

Introduction to Plant Propagation

• Asexual reproduction by Tissue Culture

• One or few cells – excised from parent plant – placed in a media containing nutrients

• Cells will divide to produce – Callus

• Callus – produces stems, roots and leaves

Plant tissues of interest to Biotechnologists

• Meristematic tissue• Are regions of cell

division – dividing and differentiating

• Tissue culture uses parts – meristematic tissues

• W/o – no plant tissues or organs can be produced

Where are meristems present?

• Found in growing tips of plant

• Root tips, Shoot tips, branch tips, flower and leaf buds

• Vascular cambium – Width of plant

Cloning of Plant Tissue Culture

• Few cells or piece of plant are grown in sterile media – agar

• Sugar, vitamins and hormones

• Optimum growing conditions• Differentiation into shoots, leaves and roots

of a new plant

Cloning of Plant Tissue Culture

• Shoots and roots are growing – transplanted into soil or another medium

• Transplanted PTC – humidity must be high- to avoid dehydration

Hormone function in plants

• Hormones/Plant growth regulators – regulate seed germination + growth of shoots, leaves, roots and flowers

• Produced in root and shoot meristems

• Two groups – auxins and cytokinins

Hormone function in plants

• Auxin – produced in shoot tips –IAA

• Causes cell elongation, leaf development and root tip elongation

• Synthetic auxins – 1-naphthaleneacetamide, 2,4-D herbicide (death of plants)

Hormone function in plants

• Cytokinins – cause cell division - Zeatin• Commercial version – Kinetin

• Ratio of auxin to cytokinin important in both stem and root elongation

• Ethylene – controls fruit ripening and leaf development

Hormone function in plants

• Gibberellin – seed, leaf bud germination, stem elongation and leaf development

• Abscisic acid (ABA) – regulates bud development and seed dormancy

• Phytochrome – pigment controls flowering

Advantages of PTC

• More plantlets in less time

• Clones of parents with no unwanted variant offspring

• Plants difficult to be grown by seed germination or stem or leaf cuttings

• Example – orchids takes five years

Factors to consider in PTC

• The species and variety of plant material

• The media and media ingredients

• Preparation of plant samples, media and equipment (sterility and temperatures)

• Done in laminar flow hoods

Plant Genetic Engineering

• Agrobacterium tumefaciens – crown gall disease in plants

• Ti plasmid (tumor -inducing) has t-DNA (transfer DNA)

• Tumor inducing genes and growth hormones on tDNA

Plant Genetic Engineering

• Ti crown gall inducing plasmid is 200 Kb in length

• Bacterial plasmid – 5-8 Kb• Scientists remove oncogenes and replace

with useful genes – genetic engineering• Virulence genes (vir) controls tranformation- Directs transfer of t-DNA from plasmid into

plant chromosomal DNA

Genetically engineered Ti plasmid

• pBI121 – Reporter genes

• GUS gene codes for ß-glucuronidase

• Breaks down carbohydrates

• 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc)- blue precipitate

Agrobacterium tumefaciens

• In plant transformation – bacteria do not enter into target cell

• Stay outside and insert Ti plasmid

• Incorporated into chromosomal DNA

• Transcription and Translation

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