Organogenesis and encapsulation of in vitro-derived propagules of Carrizo citrange [Citrus sinensis (L.) Osb. x Poncirius trifoliata (L.) Raf]

Due to widespread polyembryony, Citrus rootstocks are usually propagated by open-pollinated seed germination, although micropropagation offers many advantages. Encapsulation technology has recently attracted the interest of researchers in the field of plant propagation because it combines the advantages of zygotic or gamic seeds with those of micropropagation. In this study, we examined the encapsulation of Carrizo citrange uninodal microcuttings (3-4 mm long) and evaluated the influence of the calcium alginate coating, a short time storage at cold temperature, and different sowing substrates on the viability and regrowth of the explants. A secondary aim was to develop an efficient protocol to induce root formation in the microcuttings. The results showed that encapsulation did not negatively affect the viability, providing a satisfactory regrowth, and storage potential for 30 days at low temperature. No differences in viability and regrowth were detected between the two different sowing substrates tested (agar-solidified medium and paper filter). To optimize the production of microcuttings required to perform the encapsulation experiments, in a preliminary experiment we assessed different factors affecting the in vitro shoot regeneration from epicotyl segments (obtained from seeds of Carrizo citrange germinated in vitro), including the influence of in vitro organogenesis, explant orientation, cut surface contact with the medium, treatments with different growth regulators, and distance of the organogenic explants from the cotyledonary node. The highest organogenic response was obtained from segments horizontally cultured (particularly from the basal portion), from segments with the cut surface in contact with the medium and from explants cultured on medium supplemented with 6-benzyladenine. No significant difference in regeneration efficiency was found in response to the distance of the epicotyl portions from the cotyledonary node.