Development of explants potentially suitable for cryopreservation of the recalcitrant-seeded species Theobroma cacao L. and Barringtonia racemosa (L.) roxb.
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The two species investigated in this study were Theobroma cacao and Barringtonia racemosa. Theobroma cacao has worldwide economic importance, as cocoa (the main ingredient in chocolate) is produced from the seeds of this tree; while B. racemosa has several applications in herbal medicine. The seeds of both T. cacao and B. racemosa are highly recalcitrant and therefore not amenable to storage for any significant periods. The long-term conservation of the germplasm of these species may only be feasible via cryopreservation. The aims of the present study were to: 1) optimize in vitro regeneration protocols for different types of explants that have the potential to be cryopreserved while maintaining the genetic integrity of these two species; and 2) develop cryopreservation protocols for selected explants. For T. cacao, protocols were established for bud-break and multiplication for both in vitro - and greenhouse-derived nodal explants, as well as a rooting medium for shoots derived from axillary buds. Parameters investigated towards the cryopreservation of axillary shoots, from greenhouse nodal segments, and nodal segments from in vitro plantlets, included the size of the explant and pre-treatments for cryopreservation. Nodal segments (6 - 7 mm) and axillary shoots (2 - 4 mm) needed to be soaked in 0.5% (w/v) ascorbic acid for 10 min to minimise phenolic production and subsequent tissue death, and surface-sterilized by soaking in 1% Ca(OCl)2 solution for 5 min to reduce microbial contamination. Subsequent cryopreservation attempts involved only in vitro nodal segments because of the lack of success in achieving elongation of excised axillary buds. Vitrification and slow freezing methods, with or without the application of cryoprotectants, did not achieve successful cryopreservation. Attempts to establish a protocol for producing somatic embryos, as an alternate to axillary shoots and in vitro nodal segments, resulted in the production of globular embryogenic callus for both leaf and cotyledon explants. Cryopreservation of these explants was not investigated in the scope of this study. The study on B. racemosa focused on the development of a somatic embryogenesis protocol. Segments of embryonic axes produced globular-stage embryos when placed on MS medium supplemented with 30 g 1-1 sucrose, 1.0 g 1-1 casein hydrolysate, 2.0 mg 1-1 2,4-D, 0.1 mg 1-1 BAP and 8.0 g 1-1 agar. Various strategies were employed to obtain embryo germination, which included 1) different time intervals on callus initiation medium; 2) the use of different auxins (IAA, NAA and 2,4-D) in combination with the cytokinins BAP and kinetin; 3) desiccation and 4) cold treatments. Although somatic embryo germination was not achieved, globular embryos proceeded with development to the cotyledonary stage when cold-treated for 8 h at 4°C. This study provides some fundamental bases for further investigation towards achieving long-term conservation for both T. cacao and B. racemosa. However, the use of meristems as explants for cryopreservation is suggested to be the way forward for the cryopreservation of both species.