Optimized Auxin and Cytokinin Interactions Enable Direct Somatic Embryogenesis in the Peach Rootstock 'Guardian^®'...

Fruit tree rootstock breeding is prolonged by extended juvenile phases, high heterozygosity, limited germplasm diversity, and hybrid incompatibilities, often requiring four decades to release new cultivars. Direct somatic embryogenesis (DSE) in established peach rootstocks presents a promising avenue for rapid genetic transformation and breeding. However, peach is highly recalcitrant to in vitro regeneration, posing major challenges for organogenesis and somatic embryogenesis (SE). This study evaluated the effects of 2,4-dichlorophenoxyacetic acid (2,4-D) and Kinetin (KIN) on SE %, SE productivity, and callus % rate in the widely used Guardian^® peach rootstock. A 5 × 3 full factorial completely randomized design was used to test 15 different combinations of 2,4-D and KIN on immature cotyledons, classified as upper or lower based on their position on the preculture medium. Media formulation containing a higher concentration (3.2 µM) of 2,4-D and KIN induced SE in ~50% of lower and ~85% of upper cotyledons. Optimal SE productivity occurred with higher KIN (3.2 µM) and reduced 2,4-D (2.6 µM). Callus formation peaked with 1.8 µM 2,4-D and 3.2 µM KIN. This highly reproducible research establishes a robust whole plant regeneration system via DSE in Guardian^® peach rootstock using immature cotyledons, providing a foundation for expedited trait manipulation through biotechnological approaches.