Seed Processing

Genetically modified organisms (GMOs) Several countries have established regulatory frameworks for importing and handling GMOs. These are largely based on the Cartagena Protocol on Biosafety (CPB) which came into force in September 2003 (see www.biodiv.org/biosafety/default.aspx). National biosafety authorities, in collaboration with phytosanitary authorities, are responsible for issuing import permits, conducting risk assessments and enforcing biosafety guidelines. Researchers wishing to import GMOs must submit an application providing: details of the genetic material to be introduced; accompanying information on research and testing of the GMOs in question; and a plan for safety measures to be followed during ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

introduction. Approval is issued if the national authority determines that the GMO or GMO product poses no risk to the environment, biological diversity or human health. Prior to export of GMOs, authorization from the exporting country’s biosafety authority is also required. No authorization for export will be given if the exporting country bans the GMO or GMO product. 121 Manual of Seed Handling ANNEX II Serological methods for detecting plant pathogens General procedure using antigen-coated plate (ACP)-ELISA 1. Harvest fresh samples of leaf from samples and controls and weigh 0.2 g of each. Grind each sample in 2 ml coating buffer (0.05 M carbonate buffer)+2% w/v polyvinyl pyridine and 0.2% w/v Na2SO3. Transfer the sap to a labelled Eppendorf tube. Spin for five ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

minutes at 10 000 rpm. 2. Label the microtitre plate and load the samples, 100 µl per well. Cover the plate with parafilm and incubate at 4ºC overnight. Weigh 4 g of the healthy sample and grind in 10 ml phosphate-buffered saline solution with Tween (PBST), which acts as a blocking solution. It is made by dissolving 80.0 g NaCl, 2.0 g KH2PO4, 11.0 g Na2HPO4 and 2.0 g KCl in 2000 ml distilled water, adjusting the pH to 7.4 and adding 5.0 ml Tween 20 to make up to 10 l. Weigh the samples and dissolve. Filter through cotton wool and make the filtrate up to 80 ml. Use this to make dilutions of the antiserum. Allow to absorb at 4°C overnight. 3. Make a 0.1% solution of bovine serum albumen in PBST. 4. Rinse the plate in a gentle stream of tap water and wash three times with PBST. 5. Add PBST to all wells of the plate, 150 µl per well. Cover the plate with parafilm and incubate for 30 minutes at room temperature. 6. Completely empty the blocking solution from the plate. Without washing, add the diluted antiserum; use 100 µl per well. Cover the plate with parafilm and incubate three to four hours at room temperature. 7. Make the dilution of the alkaline phosphatase enzyme conjugate in PBST (1/1000 made fresh). 8. Wash the plate three times with PBST, then add the diluted conjugate according to the pattern on the loading diagram, starting with the most diluted. ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

Use 100 µl per well. Cover the plate with parafilm and incubate at 4°C overnight. 9. Wash the plate with PBST three times. 10. Dissolve the substrate tablet (p-NPP, Sigma) in substrate buffer (1mg/ml) (diethanolamine 10%, pH 9.8, stored in a refrigerator). 11. Add the dissolved substrate to all wells, 150 µl per well. Incubate at room temperature for 30 minutes. Evaluate and score the colour intensity of each well with the ELISA reader. 122 Handbooks for Genebanks No. 8 General procedure using tissue-blot immunoassay (TBIA) on nitrocellulose membranes 1. Collect tissues (leaves, ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

petioles, stems, etc.). 2. For thin tissues such as leaves, roll them into a tight core. For batch samples, bind them together with parafilm. 3. Cut a piece of nitrocellulose membrane (NCM), cut the top left corner and mark a grid with in pen. 4. Hold tissues in one hand and cut with a new razor blade in a steady motion with the other hand to obtain a single-cut surface. 5. Press the newly cut surface onto on grid square of the NCM with a firm but gentle force. Document what material has been blotted into which grid on a prepared form. Continue to blot leaves until all grids are full. 6. Wash the NCM three times with PBST at five-minute intervals. 7. Dilute the antiserum in healthy sap in PBST (dilution 1/500– 1/2000) and allow to absorb at room temperature for two hours or 4ºC overnight. 8. Block the NCM in 2 µg/ml polyvinyl alcohol in PBST and incubate one minute at room temperature. 9. Wash as in step 6. 10. Add diluted antiserum and incubate for one hour at room temperature. 11. Wash as in step 6. 12. Add anti-rabbit conjugate (dilution 1/1000–1/5000 in conjugate buffer) and incubate for one hour at room temperature. 13. Wash as in step 6. 14. Add substrate solution ( Seed Processing, Seed Cleaning, Stone separator, Combo Cleaner, Grading Machine, Gravity Separator  )

 (NBT/BCIP). 15. To stop the reaction, wash with de-ionized water. 123 Manual of Seed Handling ANNEX III Glossary of terms Absolute humidity: The amount of water vapour present in a unit volume of air, usually expressed in kilograms per cubic meter.