Unfertilized ovary: a novel explant for coconut (Cocos nucifera L.) somatic embryogenesis

Abstract

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 microM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 microM abscisic acid, followed by plant regeneration medium (with 5 microM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.

Genetic Fidelity Testing Using SSR Marker Assay Confirms Trueness to Type of Micropropagated Coconut (Cocos nucifera L.) Plantlets Derived from Unfertilized Ovaries

“…The crushed explants were cultured in 15 mL of standard callus induction, CRI 72 medium [2] supplemented with 160 µM 2,4-dichlorophenoxycitic acid (2,4-D), 9 µM thidiazuron (TDZ), 0.1% (w/v) activated charcoal (Haycarb, Sri Lanka), 4% sucrose [3]. The pH of the medium was adjusted to 5.8 before solidifying with 0.25% (w/v) phytagel.…”

“…CRIC65 is a cross between Sri Lanka Green Dwarf and Sri Lanka Tall coconuts while CRISL98 is a cross between Sri Lanka Tall and San Ramon Tall from the Philippines. Ovary derived plantlets were raised using the method described by Perera et al [3], with slight modifications. Unfertilized ovaries were excised from female flowers of -4 stage (considering the most recently opened inflorescences as 0 stage, the next inflorescences to open was referred as -1 and inflorescence that would open in four months later was considered as -4 maturity stage) in adult coconut palms of the two hybrids.…”

“…The interest in using tissue culture techniques for propagation of coconut has been significantly increased with the aim of developing a reliable clonal propagation method for both research purposes and commercial use. Recently, unfertilized ovary was identified as a more reliable explant for coconut micropropagation through somatic embryogenesis [3]. Even though the number of clonal plants obtained still remains low for commercial scale there is a green light with the continuous production of plantlets even at a low rate [4].…”

Somatic Embryogenesis in Recalcitrant Plants

“…However, although it allows the propagation of the progenie of known selected palms, it cannot be used for the cloning of palm individuals with known desirable agronomic traits. On the other hand, the recent developments to obtain embryogenic callus and somatic embryos from immature ovary and anther explants (Perera et al, 2007;2008;2009), provide an opportunity to try to use these calli as a source of explants (the embryogenic structures) an integrate them into the callus multiplication scheme used with plumule explants. This has already been attempted in CICY using also floral tissue explants, but in this case rachillae slices from immature inflorescences (Oropeza and Chan, unpublished results).…”

Biology, propagation and utilization of elite coconut varieties (makapuno and aromatics)

“…However, it is worth noting that during the past three decades many efforts have been undertaken to optimise the procedure for rapid multiplication of the normal coconut genotype via SE (Reviewed in [2]). Successful and consistent plantlet production of normal coconut through SE has been achieved using plumule [81], unfertilized ovary [82], and immature inflorescences [83] as the explant. Among these potential explants, the plumule is ideal for multiplication of makapuno M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT through SE.…”