Somatic embryogenesis, encapsulation, cold storage, and growth of hybrid <em>Citrus</em> [<em>C.</em> <em>paradisi</em> Macf. (‘Duncan’) × <em>C. reticulata</em> Blanco. (‘Dancy’)] shoot tip segments

Abstract

Artificial seed or encapsulation technology presents an innovative tool in plant biotechnology for an efficient and cost-effective large-scale propagation, breeding, <em>in vitro</em> conservation, non-embryonic synthetic seed production and germplasm exchange and distribution. Somatic embryogenesis is a proper method for micropropagation of fruit crops like <em>Citrus</em> and has the potential of commercial mass propagation. This method offers advantages in improving fruit species over other <em>in vitro</em> propagation methods. The combination of somatic embryogenesis and conservation strategies allows to develop both <em>in vitro</em> propagation and propagated germplasm exchange. In the first study, seeds of Minneola tangelo [<em>C.</em> <em>paradisi</em> Macf. (‘Duncan’) × <em>C. reticulate</em> Blanco. (‘Dancy’)] were placed on MS (Murashige and Skoog) medium supplemented with 500 mg l^-1 malt extract. Shoot tips from <em>in vitro</em> grown seedlings were excised and cultured on MS basal medium supplemented with 10 mg l^-1 BAP and 1 mg l^-1 napthalene acetic acid (NAA) for proliferation. A large number of micro-shoots were obtained when shoot tip explants were cultured on this medium. <em>In vitro</em> derived shoot tips were dipped in 3%, 4% and 5% of Na-alginate dissolved either in liquid MS medium supplemented with 50 g l^-1 sucrose + 10 mg l^-1 6 benzyl amino purine (BAP) + 1 mg l^-1 NAA, liquid MS medium supplemented with 50 g l^-1 sucrose and distilled water with 50 g l^-1 sucrose as treatments. The suitable artificial or synthetic seeds were formed when the micro-shoots were encapsulated in 4% Na-alginate with 100 mM CaCl₂.2H₂O. Encapsulated and non-encapsulated shoot tips were stored at 4°C for different storage periods (1-8 weeks). Maximum viability (55% after 3 weeks, 34% after 4 weeks and 12% after 7 weeks) was obtained in shoot tips encapsulated on liquid MS medium supplemented with 50 g l^-1 sucrose + 10 mg l^-1 BAP + 1 mg l^-1 NAA, while non-encapsulated shoot tips and shoot tips encapsulated on distilled water with 50 g l^-1 sucrose lost their viability completely after 3 weeks. After the storage time, encapsulated shoot tips were germinated and multiplied on MS medium containing 10 mg l^-1 BAP + 1 mg l^-1 NAA as regeneration medium. Roots were formed on the basis of shoots when they grew on MS medium enriched with 50 mg l^-1 indole-3-butyric acid (IBA). In the second study, the effects of various media on callus induction and direct and indirect somatic embryogenesis of immature seeds of <em>Citrus</em> were examined. Maximum somatic embryogenesis (direct and indirect), induction of embryonic callus and embryo maturation were achieved in MS medium supplemented with 500 mg l^-1 malt extract, 50 g l^-1 sucrose and 3 mg l^-1 BAP.