Oil palm, a tropical plant with an economic life of 20-25 years, is on high demand since its oil (palm oil) is now considered to be the world's most consumed oil. Despite the high potential for the use of clonal materials, the tissue culture technique for oil palm is difficult and laborious. One of the key steps of the process is the conversion of polyembroids into plantlets. Gelling agent has been implicated to play a role in ensuring the conversion of oil palm polyembryoids into complete plantlets. In the present study, for the first time, we report the effects of two types of common gelling agents, Agar Type 900 and Gelrite®, for enhanced conversion of oil palm polyembryoids into plantlets. Polyembryoids, developed from embryonic calli, were cultured and incubated on Murashige and Skoog semisolid media supplemented with Agar (Type 900) at 8-12 g/l or gellan gum (Gelrite®) 1.5-3.5 g/l. The effects of gelling agents on polyembryoid conversion was assessed based on the percentages of viability, survival, and polyembryoids that swelled, enlarged, and turned green, as well as on the basis of morphological characteristics, viz, number of shoots, leaves, roots, secondary somatic embryos, and callus formation. Based on the results of this study, in comparison to Agar Type 900, the Gelrite® with 3.5 g/l concentration was chosen as an effective gelling agent for conversion of polyembryoids into plantlets, since it resulted in 100% survival with 53.3% completely developed plantlets (multiple shoots with roots). The successful conversion of polyembryoids into plantlets achieved in this study, using the optimized gelling agent could be useful for pre-storage or post-storage conversion in many other plant species as well.
In the present study, polyembryoids of oil palm (Elaeis guineensis Jacq.) were cryopreserved with successful revival of 68 % for the first time using the droplet vitrification technique. Excised polyembryoids (3-5-mm diameter) from 3-month-old in vitro cultures were pre-cultured for 12 h in liquid Murashige and Skoog medium supplemented with 0.5 M sucrose. The polyembryoids were osmoprotected in loading solution [10% (w/v) dimethyl sulphoxide (DMSO) plus 0.7 M sucrose] for 30 min at room temperature and then placed on aluminium strips where they were individually drenched in chilled droplets of vitrification solution (PVS2) [30% (w/v) glycerol plus 15% (w/v) ethylene glycol (EG) plus 15% (w/v) DMSO plus 0.4 M sucrose] for 10 min. The aluminium strips were enclosed in cryovials which were then plunged quickly into liquid nitrogen and kept there for 1 h. The polyembryoids were then thawed and unloaded (using 1.2 M sucrose solution) with subsequent transfer to regeneration medium and stored in zero irradiance. Following for 10 days of storage, polyembryoids were cultured under 16 h photoperiod of 50 μmol m(-2) s(-1) photosynthetic photon flux density, at 23 ± 1 °C. Post-thaw growth recovery of 68% was recorded within 2 weeks of culture, and new shoot development was observed at 4 weeks of growth. Scanning electron microscopy revealed that successful regeneration of cryopreserved polyembryoids was related to maintenance of cellular integrity, presumably through PVS2 exposure for 10 min. The present study demonstrated that cryopreservation by droplet vitrification enhanced the regeneration percentages of oil palm in comparison with the conventional vitrification method previously reported.
The current report assesses the efficiency of encapsulation-desiccation protocol to cryopreserve oil palm (Elaeis guineensis Jacq.) polyembryoids. Specifically identified polyembryoids, comprising of haustorium and torpedo-shaped structures, were encapsulated [comprising 3% (w/v) sodium alginate and 100 mM CaCl2]. Calcium alginate-encapsulated and sucrose-precultured polyembryoids were subjected to different spans of desiccation in a laminar air-flow cabinet, followed by freezing in liquid nitrogen. The effect of sucrose preculture (with gradual exposure to 0.3, 0.5, 0.75 and 1 M for 7 days) and dehydration periods (0-10 h) under sterile air-flow on post-freezing survival and regrowth of encapsulated polyembryoids were studied. Cryopreserved and thawed polyembryoids (initially precultured in sucrose, followed by 9 h air-desiccated to 23.3% moisture content) displayed the highest survival percentage (73.3%) and regeneration (of shoot, root and secondary somatic embryo) on Murashige and Skoog regrowth medium containing sucrose (0.3-1 M) and 0.2 mg/l 2,4-dichlorophenoxy acetic acid. In addition, ultrastructural study using scanning electron microscopy exhibited successful revival of cryopreserved polyembryoids, owing to retention of cellular membrane stability through optimized and protected (encapsulated) desiccation. The present study thus substantiates the potential of this encapsulation-desiccation procedure in cryopreservation of oil palm polyembryoids for long-term conservation programs.