In previous works (Rapela, Rev. Fac. Agr. UNLP 56:17-26, 1980; ibid. 56:27-37, 1980; Plant Cell Physiol. 23:285-291, 1982; MNL 59:60-61, 1985), tests were performed on embryo and callus cultures in lysine-threonine medium in order to isolate amino acid overproducer forms of maize. Also, different explant sources, culture media and environmental conditions were tested in order to obtain embryogenic tissues with the ability to regenerate plants after several subcultures. Immature maize embryos 1.5 mm long provide an excellent material to start embryogenic cultures in media with high levels of sucrose and proline (Rapela, J. Plant Physiol. 121:119-122, 1985).
Therefore, in order to use a long-term lysine plus threonine (LT) in vitro selection system with embryogenic cultures, we developed a selection scheme with 10 steps:
1. Embryogenic callus was induced from sterilized immature maize embryos on Yu-Pei (Ku et al., 1978) medium, supplemented with 1.0 mg/l 2,4-D;120g/l sucrose and 400 mg/l proline. Thirty days in darkness at 28 C.
2. First selection cycle: the proliferating embryogenic callus was divided into small pieces (about 50 mg each) and the pieces were transferred to Petri dishes containing the modified Yu-Pei medium (step 1) along with 1 mM LT. Thirty days in darkness at 28 C.
3. Second selection cycle: medium as in step 2 but with 2.5 mM LT. Thirty days in darkness at 28 C.
4. Third selection cycle: medium as in step 3. Thirty days in darkness at 28 C.
5. Live callus showing embryogenic aspect was transferred to the initial medium (step 1) without LT. Forty five days in darkness at 28 C.
6. Fourth selection cycle: medium as in step 3. Thirty days in darkness at 28 C.
7. First regeneration cycle: embryogenic calli were transferred to a regeneration medium for plant regeneration. The culture medium was modified Murashige and Skoog (1962) (Green, Hort. Sci. 12:131-134, 1977) supplemented with 20 g/l sucrose. Sixteen hours light and 8 hours dark cycle. Light intensity was 1000 lux for thirty days at 30/26 C.
8. Second regeneration cycle: medium as in step 7. Light intensity was 3000 lux for fifteen days.
9. Regenerated plants were transferred from Erlenmeyer flasks to plastic pots containing sterile vermiculite watered with 1/4 x MS salts, and maintained in a growth chamber with 16 hours light/day at 3000 lux for fifteen days.
10. Well-established plants were moved to soil.
Approximately 200 immature maize embryos were used to start embryogenic cultures. During the selection and non-selection cycles more than 1000 calli were screened for LT resistance and embryogenic aspect.
Seventeen plantlets were regenerated from LT-resistant embryogenic calli. Six plants did not survive transplantation to vermiculite in the growth chamber. Five plants did not survive transplantation to soil in the greenhouse. One plant did not produce either flowers or ear. The remaining five plants, two of floury-a inbred genotype and three of BP normal red flint inbred genotype, each developed a single normal ear, but without flowers. These plants were pollinated in order to recover F1 progeny for further study. The F1 seeds were planted in the field during September '85.
Miguel Angel Rapela
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