A perforated ovule culture system improves nutrient accessibility and accelerates embryo rescue in cowpea

Cowpea ( Vigna unguiculata (L.) Walp., 2 n  = 2 x  = 22) is a major staple crop whose improvement is constrained by insect pests and diseases. Although wild relatives harbour valuable resistance genes, interspecific hybridization is limited by post-zygotic barriers that cause embryo abortion. Here, we developed and optimized an in vitro ovule culture system combined with ovule perforation to enhance the recovery of immature embryos in cowpea. Three genotypes, IT97K–499–35, IT86D–1010, and Sasaque, were evaluated across six MSB5– and NNB5–based medium formulations using liquid and solid culture systems, both intact and perforated ovules, and ovules collected at 1–8 days after pollination (DAP). Embryo stage, culture medium state and formulation, ovule treatment, and genotype all significantly influenced embryo germination and plant regeneration. Sasaque and IT86D–1010 consistently outperformed IT97K–499–35. Embryos at 3 DAP (globular stage) responded the earliest to in vitro ovule culture, while those at 5–8 DAP (late–heart to bent–cotyledon stages) achieved the highest regeneration rates. Solid media, particularly M1, M3, M4, and M5, outperformed liquid media in promoting embryo growth. Ovule perforation accelerated embryogenesis, doubled to quadrupled regeneration, and released dormancy in older embryos, resulting in 60% to 87% success in all three genotypes. This optimized system provides an effective platform for embryo rescue and future interspecific hybridization using wild cowpea germplasm.