Somatic proembryo production from excised, wounded zygotic carrot embryos on hormone-free medium: evaluation of the effects of pH, ethylene and activated charcoal.

Wounded zygotic embryos of cultivated carrot produce somatic proembryos on hormone-free nutrient medium containing 1 mM NH 4 (+) as the sole nitrogen source. Continued maintenance of proembryos on this medium leads to a "pure" culture of preglobular stage proembryos (PGSPs). Ethylene had no effect on this process. Also, somatic embryo production was not affected by growing cultures on activated charcoal-impregnated filter papers. However, somatic proembryos initiated on activated charcoal papers were not maintainable as PGSPs and developed into later embryo stages. Normally, medium pH dropped from 5.7 to 4 during each subculture period, but when using activated charcoal papers the pH endpoint was around 6-7 due to a leachable substance(s) within the filter papers. When powdered, activated charcoal was used in the medium as an adsorbent of products potentially released after wounding, pH dropped at the normal rate and to the expected levels; proembryos did not mature into later embryo stages and were maintainable exclusively as PGSPs. Low pH (≈ 4) is detrimental to proembryo production, but is essential to maintaining PGSPs on hormone-free nutrient medium, whereas a sustained pH >5.7 allows continued development of PGSPs into later embryo stages.