Desiccation and freezing tolerance of embryonic axes and lateral buds of Citrus spp: Implications for germplasm conservation

The objective of this research was to develop cryopreservation protocols for embryonic axes and lateral buds of ‘Pineapple’ sweet orange (Citrus sinensis [L.] Osb.) and embryonic axes of Citrus limon Burm. F. and Citrus reticulata Blanco.; Embryonic axes of three Citrus species could be desiccated to low water contents and cryopreserved in liquid nitrogen attaining high recovery percentages. Reduction of freezable water content was critical for axes survival following exposure to liquid nitrogen. Lemon seedling recovery was (63%) when axes were pre-cultured with 0.8M sucrose plus 0.5 M glycerol and desiccated to 0.15 g H₂O.g-1 dry mass. Mandarin axes pre-cultured with sucrose combined with proline and glycerol had 86% seedling recovery after cryopreservation. Sugar analysis showed that axes did not take up sucrose from the pre-culture medium. However it was found that sucrose and fructose content increased and raffinose and stachyose content decreased in axes desiccated to 0.20 mg H₂O.mg-1 dry mass. DSC analysis revealed the presence of a broad melting peak in fully hydrated axes; the size of the melting peak reduced as water was removed and minimum melting of water was observed at the point axes survived cryopreservation. The presence of glass transition upon warming was not critical for axes survival after cryopreservation. Encapsulation did not improve desiccation and freezing tolerance of embryonic axes. Pre-culture with sucrose slowed axes dehydration down.; Lateral buds were extensively damaged by desiccation and did not survive exposure to liquid nitrogen regardless of the pre-treatments tested. However, cold acclimated buds survived sub-zero temperatures as low as –13°C when cooled slowly. Leakage results indicate that cell membranes were damaged by desiccation, however these results cannot be used to predict desiccation tolerance and shoot recovery of lateral buds.; In conclusion, embryonic axes of three Citrus species can tolerate desiccation to low water contents and exposure to liquid nitrogen attaining high recovery percentages. Lateral buds were extensively damaged by desiccation and did not survive exposure to liquid nitrogen regardless of the pre-treatments tested.