Table 1.
Information on lines and treatments used in each experiment.
| Experiment I | Experiment II | Experiment III | Experiment IV | |
|---|---|---|---|---|
| Cell line | 1,198 | 1,198 | 1,247 | 1,130 |
| 2,816 | 4,089 | 1,266 | 1,375 | |
| 3,416 | 4,146 | 1,430 | 2,086 | |
| 4,611 | 5,115 | 1,810 | 2,816 | |
| 5,115 | 1,825 | 5,137 | ||
| 2,020 | ||||
| 2,064 | ||||
| 3,129 | ||||
| 4,312 | ||||
| Inoculum | 1,000–1,200 mg whole ET clumps | 1,000–1,200 mg whole ET clumps | 1,000–1,200 mg selected fresh ET | 800 to 1,050 mg whole ET clumps |
| No. replicates in proliferation | 2 in suspension, 2 in semisolid media | 2 | 1 | 3 |
| Treatments | 1x PGR | 1x PGR | 1x PGR + Rinse | 1x PGR + Rinse |
| semisolid plates (proliferation and maturation) | 0.5x PGR | 0.5x PGR + Rinse | 0.1x PGR + Rinse | |
| 1x PGR + rinse | 1x PGR + rinse + FA | |||
| Semisolid plates (ET for maturation) | semisolid plates (ET for maturation) | semisolid plates (ET for maturation) | ||
| Inoculum in maturation | Suspension pipetted; tissue amount estimated visually | Suspension pipetted; tissue amount estimated visually | Suspension pipetted; tissue amount estimated visually | 140–160 mg of tissue weighed from suspension |
| Replicates in maturation | 6 from suspensions and 6 from semisolid media | 6 from suspensions and 5 from semisolid plates | 6 from suspension bottles and 6 from semisolid plates | 9 from suspensions and 5 from semisolid plates |
| Embryos in cold storage (months) | 10 | 9 | 4 | 7 |
| Transplantation date | 18th July 2020 | 18th July 2020 | 18th July 2020 | 1st July 2021 |
In Experiment I, proliferation in suspension was compared with proliferation on semisolid media. In Experiment II, rinsing ET before maturation or halved plant growth regulator (PGR) concentration in proliferation media was implemented to improve the maturation result. In Experiment III, embryogenic tissue (ET) was grown with halved PGR and rinsed. In Experiment IV, ultralow PGR concentration with rinsing the ET was tested. Increased gas exchange was also tried for a 1x PGR suspension (Forced aeration, FA). Semisolid grown ET was used as a control for maturations in all experiments. 1x PGR (10 μM 2,4-D and 5 μM BA) was the control treatment; experimental treatments with modified PGR rates (0.5x and 0.1x) were applied in Experiments II to IV. Additionally, ETs were rinsed (+ rinse) to reduce PGR residues during maturation. In Experiments I–III, ET was pipetted from suspension onto filter papers, and in Experiment IV, ET was matured as if from semisolid media by weighing 140 to 160 mg from suspension-grown ET dried with suction.