Plant tissue culture is an important and continually emerging field applied to improve the production, propagation, and conservation of cultivated and wild plant species. This Special Issue of Horticulturae belongs to the section "Propagation and Seeds" and is dedicated to recent developments in in vitro tissue culture technologies and their use in micropropagation, in vitro plant regeneration, and the germplasm conservation of horticultural crops. The papers in this Reprint have been peer reviewed by experts and guest editors and published in Horticulturae in full open access. This Special Issue contains eleven publications, including nine research articles, one review, and one essay article, which provide a representative and valuable collection of the in vitro tissue culture technologies used for horticultural species. It also provides perspectives for future studies and offers a diverse learning experience appropriate for students, technicians, and scientists of plant sciences and plant/agricultural biotechnology.