1. Laboratory Exercise – Seed germination of Bean and Corn
Plant Tissue Culture
Laboratory Exercise – Seed germination of Bean and Corn

2. 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
To gain identical to original – Asexual reproduction. Not new varieties always – but clones or identical plants are needed

3. 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
Different kinds of as methods that scientists use to create clones.

4. 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
Resulting plant is identical to plant that was source of original plant. Maintained in sterile conditions therefore no pest problems

5. 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

6. 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

7. 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
Growing conditions in tubes, under fluorescent lights at appropriate temperature. First evidence of activity is swelling of explant – is a result of large number of cell divisions

9. 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

10. 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
Cause shoot and root growth – based on their concentration

11. 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)

12. 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

13. 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

14. 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

15. 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
Bacteria and mold easily grow

16. 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

17. 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

18. 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

20. 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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