Linkage mapping and QTL analysis in coconut (Cocos nucifera L.)

Different DNA marker types were used to construct linkage maps in coconut (Cocos nucifera L.; 2n = 32) for the two parents of the cross Malayan Yellow Dwarf (MYD) × Laguna Tall (LAGT). A total of 382 markers was sufficient to generate 16 linkage groups for each parent. The total genome length corresponded to 2226 cM for the LAGT map and 1266 cM for the MYD map with 4–32 markers per linkage group. Common markers allowed the association of 9 linkage groups for the two parents MYD and LAGT. QTL analysis for the trait early germination identified six loci. These QTLs correlate with early flowering and yield, representing characters which are important in coconut breeding. The co-segregation of markers with these QTLs provides the first opportunity for marker-assisted selection in coconut breeding programmes. Received: 22 September 1999 / Accepted: 29 November 1999     

Androgenic potential in coconut (Cocos nucifera L.)

Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38°C) or cold (4°C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38°C for 6 days. Modified Eeuwens Y₃ liquid medium supplemented with 100 μM 2,4-dichlorophenoxyacetic acid (2,4-d), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y₃ medium containing 66 μM 2,4-d, followed by transfer to Y₃ medium without plant growth regulators and finally to Y₃ medium containing 5 μM 6-benzyladenine (BA) and 0.35 μM gibberellic acid (GA₃), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthers.     

Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) in vitro

Complete plants were grown from zygotic embryos cultured on Y3 basal liquid medium supplemented with coconut milk, BA and NAA. Explants from stem, leaf and rachilla of mature coconut trees turned green and swelled on Y3 semi-solid basal media supplemented with 2,4-D, K, NAA, BA and activated charcoal. Callus was initiated in explants from the subapical regions of the stem on Y3 basal medium supplemented with 2,4-D (4.52×10²M). Globular embryo-like structures were obtained when this callus was subcultured to auxinless medium. Root formation was obtained from leaf explants on Y3 basal medium containing citric acid, ascorbic acid and 2,4-D (4.52×10² M). Globular embryo-like structures were also obtained directly from leaf explants on a Y3 basal medium supplemented with 2,4-D (2.26×10² M). Callus isolated from rachilla explants on Y3 basal medium containing 2,4-D(4.52×10² M), formed nodular structures when transferred to medium with 2,4-D (2.3×10¹ M). These nodules developed roots from the base of the nodular growth whereas from the upper portion shoots were observed on Y3 basal liquid medium.Abbreviations K kinetin - BA Benzyl adenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - CM Coconut milk - IAA Indole acetic acid - 2iP N⁶-r-r-dimethyl allyl amino purine NCL Communication No. 3471     

Dehydration improves cryopreservation of coconut (Cocos nucifera L.)

Cryopreservation of coconut can be used as a strategy to back up the establishment of living collections which are expensive to maintain and are under constant threat from biotic and abiotic factors. Unfortunately, cryopreservation protocols still need to be developed that are capable of producing a sizeable number of field-grown plants. Therefore, we report on the development of an improved cryopreservation protocol which can be used on a wide range of coconut cultivars. The cryopreservation of zygotic embryos and their recovery to soil-growing plants was achieved through the application of four optimised steps viz.: (i) rapid dehydration; (ii) rapid cooling; (iii) rapid warming and recovery in vitro and (iv) acclimatisation and soil-supported growth. The thermal properties of water within the embryos were monitored using differential scanning calorimetry (DSC) in order to ensure that the freezable component was kept to a minimum. The feasibility of the protocol was assessed using the Malayan Yellow Dwarf (MYD) cultivar in Australia and then tested on a range of cultivars which were freshly harvested and studied in Indonesia. The most efficient protocol was one based on an 8-h rapid dehydration step followed by rapid cooling step. Best recovery percentages were obtained when a rapid warming step and an optimised in vitro culture step were used. Following this protocol, 20% (when cryopreserved 12 days after harvesting) and 40% (when cryopreserved at the time of harvest) of all MYD embryos cryopreserved could be returned to normal seedlings growing in soil. DSC showed that this protocol induced a drop in embryo fresh weight to 19% and significantly reduced the amount of water remaining that could produce ice crystals (0.1%). Of the 20 cultivars tested, 16 were found to produce between 10% and 40% normal seedlings while four cultivars generated between 0% and 10% normal seedlings after cryopreservation. This new protocol is applicable to a wide range of coconut cultivars and is useful for the routine cryopreservation of coconut genetic resources.     

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.)

Molecular Biology Reports - Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient...     

Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis

A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases. Received: 19 March 1997 /Revision received: 11 September 1997 / Accepted: 4 October 1997     

Independent origins of cultivated coconut (Cocos nucifera L.) in the old world tropics

As a portable source of food, water, fuel, and construction materials, the coconut (Cocos nucifera L.) played a fundamental role in human migrations and the development of civilization across the humid tropics. Here we investigated the coconut's domestication history and its population genetic structure as it relates to human dispersal patterns. A sample of 1,322 coconut accessions, representing the geographical and phenotypic diversity of the species, was examined using ten microsatellite loci. Bayesian analyses reveal two highly genetically differentiated subpopulations that correspond to the Pacific and Indo-Atlantic oceanic basins. This pattern suggests independent origins of coconut cultivation in these two world regions, with persistent population structure on a global scale despite long-term human cultivation and dispersal. Pacific coconuts show additional genetic substructure corresponding to phenotypic and geographical subgroups; moreover, the traits that are most clearly associated with selection under human cultivation (dwarf habit, self-pollination, and "niu vai" fruit morphology) arose only in the Pacific. Coconuts that show evidence of genetic admixture between the Pacific and Indo-Atlantic groups occur primarily in the southwestern Indian Ocean. This pattern is consistent with human introductions of Pacific coconuts along the ancient Austronesian trade route connecting Madagascar to Southeast Asia. Admixture in coastal east Africa may also reflect later historic Arab trading along the Indian Ocean coastline. We propose two geographical origins of coconut cultivation: island Southeast Asia and southern margins of the Indian subcontinent.     

Population structures of Brazilian tall coconut (Cocos nucifera L.) by microsatellite markers

Coconut palms of the Tall group were introduced to Brazil from the Cape Verde Islands in 1553. The present study sought to evaluate the genetic diversity among and within Brazilian Tall coconut populations. Samples were collected of 195 trees from 10 populations. Genetic diversity was accessed by investigating 13 simple sequence repeats (SSR) loci. This provided a total of 68 alleles, ranging from 2 to 13 alleles per locus, with an average of 5.23. The mean values of gene diversity (H_e ) and observed heterozygosity (H_o ) were 0.459 and 0.443, respectively. The genetic differentiation among populations was estimated at θ^P=0.1600and the estimated apparent outcrossing rate was t_a = 0.92. Estimates of genetic distances between the populations varied from 0.034 to 0.390. Genetic distance and the corresponding clustering analysis indicate the formation of two groups. The first consists of the Baía Formosa, Georgino Avelino, and São José do Mipibu populations and the second consists of the Japoatã, Pacatuba, and Praia do Forte populations. The correlation matrix between genetic and geographic distances was positive and significant at a 1% probability. Taken together, our results suggest a spatial structuring of the genetic variability among the populations. Geographically closer populations exhibited greater similarities.     

Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.)

One prerequisite to reliable molecular biology work is that the genomic DNA of a sample be of good quality. Coconut is quite difficult to work on because of the high lipid and polysaccharide content of its endosperm and the high polyphenol content of its leaves. This study aimed to determine which protocol to use and which part of the coconut tree is most appropriate to extract good-quality genomic DNA. Genomic DNA from the solid endosperm was found to be of poor quality because of high levels of lipid and galactomannan contaminants. By using a modified protocol by Cheung et al. (1993) as modified by Rogers et al. (1996) and by Dellaporta et al. (1983) as modified by Datta et al. (1997), genomic DNA extracted from the young leaves of the first emergent frond provided enzyme-digestible, good-quality DNA. The modification involved the use of a higher salt concentration (2 M instead of 0.5 M) in the extraction buffer and the use of polyvinylpolypyrrolidone. Moreover, this modified protocol did not involve the use of organic solvents.     

Plant regeneration from cultured immature inflorescences of coconut (Cocos nucifera L.): evidence for somatic embryogenesis

Immature inflorescences of coconut belonging to three different genotypes were cultured on a solid medium supplemented with activated charcoal (2%) and a range of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (from 1.5 to 3.5 × 10^–4M). Globular white callus formed from immature floral meristems, depending on inflorescence age and 2,4-D concentration. Acquisition of embryogenic competence is described histologically. Somatic embryos presented a functional bipolar organization with a completely differentiated shoot meristem which is reported here for the first time in coconut tissue culture. Embryo maturation allowed reliable plant regeneration of this in vitro recalcitrant species. Details are given of exogenous hormonal requirements for the acquisition of embryogenic competence and embryo maturation.     

Antibiofilm activity of coconut (Cocos nucifera Linn.) husk fibre extract

In this study, antibiofilm activity of coconut husk extract (CHE) was tested by various assays in the laboratory. The effects of CHE on extracellular polymeric substance (EPS) production, hydrophobicity and adhesion ability of Pseudomonas sp., Alteromonas sp. and Gallionella sp. and the antimicrobial activity of the extract against these bacteria were assessed. CHE was found to possess antibacterial activity against all the bacterial strains and affected the EPS production. The CHE affected the growth of the biofilm-forming bacteria in a culture medium. The hydrophobicity of the bacterial cells was also changed due to the CHE treatment. The active compound of the CHE was characterised by thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and fourier transform infrared (FT-IR) analysis. HPLC spectrum showed a single peak and the FT-IR spectrum indicated the presence of an OH-group-containing compound in the extract. In conclusion the CHE could be used as a source for the isolation of antifouling compounds.     

Morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured zygotic embryos

In this study, we compared the evolution of morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured embryos and from seeds over an 8-yr period. At the end of the second year after planting, the height and root collar diameter of plants originating from in vitro cultured embryos were significantly lower than those originating from seeds. However, palms from both categories had the same number of leaves. When palms originating from in vitro plantlets and from seeds were observed later in their development, they were similar for most morphological characteristics measured, except for minor differences in inflorescence morphology, which were still present 8 yr after planting. The flowering pattern, bunch, and fruit production were similar between the two categories of palms. These results indicate that in vitro culture of zygotic embryos does not adversely affect further development of palms in natural conditions.     

QTL analysis of fruit components in the progeny of a Rennell Island Tall coconut (Cocos nucifera L.) individual

We investigated the genetic factors controlling fruit components in coconut by performing QTL analyses for fruit component weights and ratios in a segregating progeny of a Rennell Island Tall genotype. The underlying linkage map of this population was already established in a previous study, as well as QTL analyses for fruit production, which were used to complement our results. The addition of 53 new markers (mainly SSRs) led to minor amendments in the map. A total of 52 putative QTLs were identified for the 11 traits under study. Thirty-four of them were grouped in six small clusters, which probably correspond to single pleiotropic genes. Some additional QTLs located apart from these clusters also had relatively large effects on the individual traits. The QTLs for fruit component weight, endosperm humidity and fruit production were found at different locations in the genome, suggesting that efficient marker-assisted selection for yield can be achieved by selecting QTLs for the individual components. The detected QTLs descend from a genotype belonging to the “Pacific” coconut group. Based on the known molecular and phenotypic differences between “Pacific” and “Indo-Atlantic” coconuts, we suggest that a large fraction of coconut genetic diversity is still to be investigated by studying populations derived from crosses between these groups. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants

Summary Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embrogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenenis callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.     

Inter-relationships among root cec,yield and mono- and divalent cations in coconut (Cocos nucifera L.)

Plant and Soil - A study was conducted on different yield groups of coconut palm (West Coast Tall) to investigate the relationships among root CEC, yield and mono- and divalent cations. The results...     

Evaluating genetic relationships between indigenous coconut (Cocos nucifera L.) accessions from Sri Lanka by means of AFLP profiling

 PCR-based DNA profiling of coconut palms indigenous to Sri Lanka was conducted using amplified fragment length polymorphism (AFLPs). A total of 322 amplification products were generated from the 42 genotypes with eight pairs of primers (EcoRI and MseI). Overall most variation was detected in the tall (Typica) rather than the intermediate (Aurantiaca) and dwarf (Nana) forms. A hierarchical analysis of molecular variance (AMOVA) was used to quantify and partition levels of variability into between- and within-form components. This revealed that for the inbreeding dwarf and intermediate forms most variation was observed between, rather than within, forms. In contrast, the outbreeding tall forms exhibited as much variation within as between forms. These observations have important implications for the maintenance and collection of coconut germplasm. This study also provided insights into the genetic (as opposed to phenotypic) relatedness of coconut accessions. Morphologically the Aurantiaca group of accessions are considered to be intermediate between the tall and dwarf accessions. Estimation of genetic relatedness based on AFLP analysis identified the Aurantiaca group as being more similar to the dwarf rather than the tall group. In addition, putative duplicate accessions were identified in the Aurantiaca group. Information emerging from this study will facilitate the management of coconut germplasm and optimise the choice of genetically divergent parents for crossing. Received: 16 June 1997 / Accepted: 14 October 1997     

Purification and crystallization of coconut globulin cocosin from Cocos nucifera

Cocosin is a legume class reserve protein found in coconut endosperm. Using coconut endosperm, two methods of purification were done. Crystallization was achieved by vapor diffusion (hanging drop) method using MPD, PEG 3350 and PEG 4000 as precipitants. X-ray diffraction data to 3.5-A resolution were collected using Mar345 image plate detector system. Crystals of cocosin grown under 20% MPD, are rhombohedral with space group R3 and cell dimensions a=92.829 A, b=92.829 A, c=215.290 A.