Technical Protocol 1.0
Principals Of Germplasm And Disease-Indexed Line Maintenance
Once a line has been established as meeting the standards for
germplasm it can be maintained for many years if proper care is given
to details. The most common mistakes in germplasm maintenance involve
line identity, line purity and secondary testing.
Each germplasm line should have a double identity system: both an initial source number or code and a name or key identifier. Lines should be maintained in culture vessels which are contained in rigid blocks in the germplasm culture area, such as in plastic trays or racks. Each germplasm block is labeled with all relevant identification: accession number, name or breeding number, description, date of accession, etc. Accession numbers should refer to a log kept with additional relevant information (disease testing status, source or origin of the line, etc.). Each test tube or culture vessel within the block is labeled with the accession number.
Operators who are working with the germplasm do not work with more than one line in the workstation at any one time. Only a portion of the culture base is subcultured at any one time, so that a back up of older cultures is always maintained alongside the newly subcultured test tubes.
Long term line maintenance
No germplasm lines are ever derived from left-over propagation runs. Germplasm is a one-way culture base; cultures which exit germplasm can never come back in. They can go out to testing or out to production, but cultures in germplasm must not be contaminated by cultures from other sources. Germplasm should be maintained under minimal conditions, such as growth regulator free medium, to ensure the least amount of genetic change. Ideally, germplasm is maintained in a culture room or area separated from the production culture base. Also, a separate area for new lines and quarantined (untested) lines should be enforced.
Annual certification of germplasm lines
Every year a single test tube is divided into 3-4 new test tubes, 3 for testing of key viruses, bacteria, etc. and one for starting the next year's germplasm. After 6-8 weeks, when viability and test results have been confirmed, the remaining back-up test tubes in germplasm are destroyed. Annual grow-out for verification of line identity and genetic purity is also sometimes warranted. Slight changes in genetics have been well documented, especially in certain species.
Propagation or production increases starting with germplasm lines
For production a single germplasm test tube should be selected for increase. This test tube is divided and increased to meet production needs. A single test tube of this individual is kept back, either for testing or grow-out to verify the line characteristics.