Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 64

Mango embryos are recalcitrant, i.e. they cannot tolerate desiccation during maturation and they do not undergo developmental arrest. Lacking a period of developmental arrest, mango embryos develop to maturity and then germinate in an uninterrupted sequence. Mango seeds (and embryos) cannot survive for more than 3 or 4 weeks under in vitro minimal growth storage conditions (Parisot, 1988) or in a conventional seed bank. Monsalud et al. (1995) demonstrated that 4–5 mm somatic embryos (late heart stage) could be partially desiccated and stored dry in Petri dishes without plant growth medium for >30 days without any loss of viability; however, larger somatic embryos could not survive this treatment. This observation has implications for future studies that might focus on developing the concept of an ‘artificial seed’ (i.e. somatic embryo) genebank for vegetatively propagated tropical fruit trees.

ABA is associated with initiation of developmental arrest and desiccation of orthodox type seeds and embryos and is effective in vitro at relatively low concentrations for somatic embryos of the orthodox type (Bewley and Black, 1985). Pliego Alfaro et al. (1996a,b) were able to arrest late heart-stage mango somatic and nucellar embryo development for several months with high levels of ABA (minimum 100 μM) in the growth medium. ABA had a strong residual effect on mango somatic embryo growth inhibition, which persisted for c.1 month after subculture of somatic embryos onto medium without ABA. Increasing the osmolarity of the plant growth medium also inhibited somatic embryo development; however, there was no residual effect.

Cryopreservation of embryogenic cultures of mango using different procedures has been demonstrated to be feasible (Wu et al., 2003, 2014; R. Nadgauda and P. Moon, Homestead, Florida, USA, personal communication). Wu et al. (2003) compared three cryopreservation protocols for embryogenic ‘Zihua’ cultures: (i) encapsulation-dehydration; (ii) pregrowth-dehydration; and (iii) vitrification. The encapsulation-dehydration procedure was unsuccessful, and only limited survival (8.3%) was obtained following desiccation of PEMs for 1 h to 58.5% moisture content prior to freezing in liquid nitrogen. Vitrification, involving treatment of PEMs with PVS2 solution (Sakai et al., 1991) for 20 min prior to freezing in liquid nitrogen, was successful (94.3%).

Embryogenic ‘Hindi’ cultures have also been introduced into cryogenic storage (R. Nadgauda and P. Moon, Homestead, Florida, USA, personal communication), and somatic embryos have been recovered from these cultures. Two procedures were followed: (i) stepwise cooling in which cryoprotected (5% DMSO and 5% glycerol) embryogenic cultures were cooled in ‘Mr. Frosty’ containers at the rate of −1°C/min from room temperature to −75°C followed by rapid cooling to −196°C; and (ii) rapid cooling (vitrification). Following the removal of cryovials from liquid nitrogen and rapid warming to room temperature, cultures were thoroughly washed with maintenance medium and plated on semisolid maintenance medium formulation. Somatic embryo development was initiated by subculturing the PEMs on somatic embryo maturation medium.