In Vitro Plant Regeneration from Petioles of Spinach (Spinacia oleracea L.)

Spinach (Spinacia oleracea L.) is an economically important leafy vegetable crop with an annual global production of 32 million tons. However, it is vulnerable to chewing as well as sucking insects. In vitro plant regeneration can significantly contribute to efforts toward crop improvement. This study is aimed at developing a highly efficient protocol for direct in vitro regeneration of S. oleracea from five different explants (cotyledons, leaves, petioles, hypocotyls, and root segments) and five different regeneration media. Results intimated that only petioles exhibited a response for direct regeneration; hence, they were used in further experiments. More than 35% of the petioles regenerated directly into shoots, which were later separated and cultured onto a rooting medium. All other explants showed variable responses; however, none of them could regenerate in vitro. As far as the role of plant growth regulators is concerned, gibberellic acid (GA₃) appeared to be the most imperative one for direct in vitro regeneration and bringing about maximum regeneration in Murashige and Skoog (MS) medium augmented with 2 mg L^–1 gibberellic acid (GA₃), 0.4 mg L^–1 naphthalene acetic acid (NAA), and 1 mg L^–1 benzyl amino purine (BAP). The resultant plants were acclimatized in the greenhouse and resulted in healthy, fleshy leaves. For the first time, the protocol describes efficient direct in vitro regeneration from green tissues of S. oleracea. As this is an efficient in vitro regeneration system, it may be a step forward to engineer/edit the genome of this green vegetable for valuable traits including agronomic traits improvement, biofortification, and biopharmaceutical production in the future.