An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture

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Abstract
Regenerants from a 30-month-old haploid and a 10-month-old diploid tissue culture were cross-pollinated to generate a synthetic genotype (HE/89) with improved competence for maintenance of totipotency in various cultured expiants. The HE/89 zygotic embryos developed friable, embryogenic cultures in the commonly used MS-and N6-based media without the addition of L-proline. By optimalization and changing the culture conditions, we were able to regulate the maintenance of the earlier, more synchronous (Type II) and the later, asynchronous (Type I) in vitro embryogenesis, as well as the shift between different ontogenic stages. Within 70 days after the inoculation of immature embryos a relatively homogeneous, early-embryogenic suspension culture usable for protoplast isolation was established from the initially surface-grown cultures. Using modified solutions for protoplast isolation and culture, viable protoplasts were reproducibly obtained from which plants were regenerated via defined ontogenic steps. Despite the long in vitro history of the parental genotypes, 60–70% of the more than 500 plants derived from the HE/89 protoplasts set seeds following self or sib-pollination.