Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos does not induce morphological, cytological or molecular changes in recovered seedlings

Abstract

The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates; no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.

Tissue culture and associated biotechnological interventions for the improvement of coconut (Cocos nucifera L.): a review

“…0.8 % agar (to be kept in dark condition for 8 weeks), b Further development of shoot and roots on an embryo cultured plantlet. c Photoautotrophic system (CO 2 enrichment growth chamber) developed to improve seedling growth, d comparison between an acclimatized plantlet grown in a CO 2 enrichment environment and one covered by conventional plastic bag, e Plumule tissue emerging from a zygotic embryo and subsequently used as initial explant for callus induction, f-g different responses in callus induction media supplemented, respectively, with 200 lM and 600 lM 2,4-D, h Maturation of somatic embryos in a reduced 2,4-D medium, i aseptic isolation of zygotic embryos for cryopreservation, j rapid dehydration of sterilized embryos using fan-forced air apparatus, before being plunged into liquid nitrogen, k-l No significant differences in the morphology observed during the development and acclimatization of plantlets derived from cryopreserved embryos and normal embryos (these two photos are reprinted from Sisunandar et al 2010a, with permission) (P plumule, GP germ pore, NES non-embryogenic structures, GES globular embryogenic structures). Bar a, e, f-5 mm; g, h-1 mm; l-5 cm Planta protocol using CO 2 enrichment (1600 lmol mol -1 ) during the light phase was found to improve seedling health, growth, and the percentage of seedlings established (Samosir and Adkins 2014) (Fig.…”

“…Regarding production of homozygous inbred lines, Perera et al (2008b) have reported the production of doubled haploid plants via anther-derived embryogenesis. Furthermore, it is now possible to cryopreserve, and then recover coconut embryos for in long-term conservation programs, without inducing morphological, cytological or molecular changes in the regenerated plants (Sisunandar et al 2010a). Although genetic transformation in coconut has been attempted Andrade-Torres et al 2011), achievements have been quite limited to date.…”

“…Coconut field cultivation faces many challenges, including the instability of the market for its traditional products. Productivity is affected by age, declining steadily after 35 years due to a decline in leaf area, by the rundown of soil nutrients, and through damage caused by cyclones, storms and tsunamis (Sisunandar et al 2010a;Samosir and Adkins 2014). Rapid spread of major pests and incurable diseases, such as phytoplasma-caused lethal yellowing and viroid-caused cadang-cadang, has resulted in a significant fall in the land area planted to coconut (Cordova et al 2003;Harrison and Jones 2003;Lee 2013).…”

Embryo Incision as a New Technique for Double Seedling Production of Indonesian Elite Coconut Type “Kopyor”

“…The Sisunandar method [12] for embryo isolation from the nut and surface sterilization of the embryos was applied with minor modifications. After cracking the nuts, the zygotic embryos ( Figure 1(A)) with a small portion of endosperm were isolated from a specific region of the endosperm using a metal spoon.…”

“…This medium then, may also be used for root induction in coconut embryo culture. The use of MS basal medium during the recovery process after incision and splitting showed that even though HEC medium is the most widely used medium for coconuts [4,[11][12][13][16][17], MS basal medium showed the best results during the recovery process of the incised embryos. Major differences in macronutrient composition between MS and HEC are the most plausible explanation for this result [18], as it is widely known that macronutrients such as nitrogen (in the form of NO 3 -and NH 4 + ) play an important role in both morphogenesis and growth.…”