Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 121

The first attempt to cryopreserve coconut tissues was undertaken with immature zygotic embryos using a chemical dehydration and slow freezing technique (Bajaj, 1984). However, attention has shifted towards using mature (11 months post-pollination) zygotic embryos (Sisunandar et al., 2014) and using a physical dehydration method or using plumular tissues excised from mature zygotic embryos and using chemical dehydration.

The cryopreservation protocol consists of four steps: (i) preculture of explant in preparation for drying; (ii) dehydration; (iii) freezing; and (iv) recovery involving thawing and plantlet regeneration. Three tissue dehydration methods have been attempted: (i) chemical dehydration; (ii) slow physical dehydration (desiccation in a laminar air flow hood); and (iii) rapid physical dehydration (fan forced drying using silica gel). For chemical dehydration sucrose, glucose and glycerol, all at high concentrations (>10%, w/v) are most commonly used. Encapsulation using sodium alginate (3%) following tissue dehydration using sucrose (5%) has also been attempted using plumule tissue (N’Nan et al., 2008). For slow physical dehydration, various drying durations (7–48 h) have been used. The recovery signs are often good, although few plantlets are produced by these methods. For rapid physical dehydration, a flash drying approach has been used (Sisunandar et al., 2010b). By following water loss using differential scanning calorimetry, drying embryos to 20% moisture content in 8 h gave the best survival after cryopreservation, giving up to 40% of cryopreserved embryos that germinate and survive in soil, a level that had not been achieved using any previous method. This cryopreservation method did not induce any measurable genetic change in the recovered plants (Sisunandar et al., 2010a).

A rapid freezing approach has been used for coconut tissues. In most cases, the dehydrated tissues are transferred into cryovials and plunged directly into liquid nitrogen. In most cases, a rapid thawing approach is used whereby the cryopreserved tissues are submerged into a water bath set at 40°C for 3 min. The selection of the correct recovery and embryo germination media has been critical for the success of cryopreservation. The MS (Murashige and Skoog, 1962), MW (Morel and Wetmore, 1951) and Y3 (Eeuwens, 1976) media formulations have all been commonly used for tissue recovery with the latter medium preferred in most studies (Sisunandar et al., 2010b, 2012; Sajini et al., 2011). It is noteworthy that the application of 2,4-D, NAA or kinetin, either alone or in combination, did not significantly enable embryo germination or plantlet recovery (Bajaj, 1984; Chin et al., 1989). On the other hand, high doses of sucrose (4–6%) have been shown to be important for the germination of the recovered embryos (N’Nan et al., 2008; Sisunandar et al., 2010b; Sajini et al., 2011). Establishment of plants in soil following cryopreservation of coconut embryos has only been reported using the chemical dehydration approach of Sajini et al. (2011) and by the physical dehydration approach of Sisunandar et al. (2010b).