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In vitro germination

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Preparing the seeds

Break the dormancy of the seeds by the following procedures:

a. For Oryza sativa - stabilize the seeds from the medium-term storage at room temperature for 2 days. Put the seeds in small packets and place them inside the oven at 50 ° for 3-5 days. Take the seeds out of the oven and stabilize at room temperature for 2 days. Remove the seeds hulls.

b. For the other Oryza species - stabilize the seeds from the medium term storage at room temperature for 2 days. Follow the dormancy breaking protocols in Section 3 of this manual.

Seeding on agar medium

(All steps in this section should be done inside a laminar flow.)

  1. Transfer the seeds from each packet to properly labeled 4” x 1” (l x d) tubes.
  2. Pour enough 70% ethanol to immerse the seeds in each tube and wash the seeds by shaking the tube several times. Pour off ethanol in a container.
  3. 3. Pour 20% sodium hypochlorite (NaOCl) into each tube. Leave this for 15 min to sterilize the seeds. Shake the tubes occasionally. After 15 min, pour off the sodium hypochlorite in a container.
  4. Pour sterile distilled water into each tube. Wash the seeds by shaking the tubes several times. Pour off the water into a container. Do this step 3 times.
  5. Transfer the seeds from each tube to petri dishes lined with sterile filter paper to blot dry them.
  6. Plant the seeds in 5” x 1” or 8” x 1” (l x d) test tubes with agar media (see Table 9-2 for media preparation; see Table 9-1 for stock solution of culture media). First, remove the tube’s cover and flame the rim of the tube. For accessions with more than 20 seeds, use a 125 ml Erlenmeyer flask. The tube or the flask should be properly labeled with the accession number.
  7. With a sterile spatula, take a seed from the petri dish. Put this seed on to the surface of the agar media. Do this for the rest of the seeds. For accessions with more than 20 seeds, transfer 10-15 seeds per flask.
  8. Flame the rim of the tube and the cover’s underside before sealing it again.
  9. Do steps 6-8 for all accessions.

Plant establishment

  1. Stand the tubes with the seeds in a test tube rack and put them inside a dark cabinet in the culture room until the shoot and root emergence. Inspect the tubes occasionally to find out if the seeds have germinated.
  2. Transfer the tubes with seedlings into the lighted shelves in the culture room. The shelves should have a 12/12 h light and dark cycle.
  3. Let the seedlings grow until they are about 8 cm long. These are now ready for transferring to the culture solution.
  4. Label each hole of a 9” x 12” styropor with the accession number of the seedlings.
  5. Put the styropor in an 11” x 14” tray filled with distilled water.
  6. Remove each seedling by scooping out the agar media with a spatula.
  7. Wash the roots of the seedling with distilled water in a tray to remove all the agar media adhering to it . Do this very carefully to avoid damaging the roots.
  8. Sandwich the lower portion of the stem (1 cm from the base) of each seedling with a 0.5” x 2” foam. Insert the foam with the seedling into the corresponding hole of the styropor.
  9. Transport the seedlings to the Phytotron. Replace the distilled water in the tray with culture solution (see Table 9-4 for culture solution preparation and Table 9-3 for the stock solution). Allow the seedling to grow inside the glass chamber (21/29 °, 70% RH) or an indoor growth cabinet (21/29 °, 70% RH, 12 h light at 900 µ¨m-2s-2) for 2-3 weeks or until the seedlings are at the 2-3 tiller stage.
  10. Maintain the pH of the culture solution daily. It should remain at pH 5.5.
  11. Replace the culture solution twice a week.
  12. Transplant the seedlings to pots in the nursery.