Laboratory Exercise – Seed germination of Bean and Corn 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 To gain identical to original – Asexual reproduction. Not new varieties always – but clones or identical plants are needed

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.

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

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 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

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 Cause shoot and root growth – based on their concentration

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 Bacteria and mold easily grow

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

This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

Disclaimer This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.