Alternative splicing of flowering time gene FT is associated with halving of time to flowering in coconut.

Coconut palm has two distinct types-"tall" and "dwarf"-which differ morphologically. Tall coconut varieties need 8-10 years to start flowering, while dwarf coconut varieties only require 3-5 years. We compared seedling and reproductive stage transcriptomes for both coconut types to determine potential molecular mechanisms underlying control of flowering time in coconut. Several key genes in the photoperiod pathway were differentially expressed between seedling and reproductive leaf samples in both tall and dwarf coconut. These genes included suppressor of overexpression of constans (SOC1), flowering locus T (FT), and Apetala 1 (AP1). Alternative splicing analysis of genes in the photoperiod pathway further revealed that the FT gene produces different transcripts in tall compared to dwarf coconut. The shorter alternative splice variant of FT [which included a 6 bp deletion, alternative 3' splicing sites (A3SS)] was found to be exclusively present in dwarf coconut varieties but absent in most tall coconut varieties. Our results provide a valuable information resource as well as suggesting a probable mechanism for differentiation of flowering time onset in coconut, providing a target for future breeding work in accelerating time to flowering in this crop species.

Protocol for the Micropropagation of Coconut from Plumule Explants.

Coconut is a crop that is economically important in several countries throughout the world. Unfortunately, production is decreasing because palms are affected by very serious phytoplasma diseases, such as lethal yellowing, and also most of coconuts are already very old. On the other hand, markets for coconut products have been rapidly growing in recent years. Hence, replanting of most cultivation surface worldwide, as well as establishing new surface, is urgently needed. This is an immense task, requiring at least a billion coconut palms that cannot be accomplished by traditional propagation through seed. Therefore the biotechnological alternative of micropropagation by somatic embryogenesis is needed. Research has been carried out on this subject in laboratories in several countries studying different approaches, testing different types of explants. The most responsive tissue has been plumule from zygotic embryos. A protocol for micropropagation of coconut based on plumule explants is described here. It involves the use of different media that are based on Y3 medium complemented with activated charcoal, gelling agent, sucrose, and growth regulators. These media allow the formation of embryogenic callus and somatic embryos, growth of shoots, and development of plantlets.

Predator performance is impaired by the presence of a second prey species.

The simultaneous infestation of a plant by several species of herbivores may affect the attractiveness of plants to the natural enemies of one of the herbivores. We studied the effect of coconut fruits infested by the pests Aceria guerreronis and Steneotarsonemus concavuscutum, which are generally found together under the coconut perianth. The predatory mite Neoseiulus baraki produced lower numbers of offspring on fruits infested with S. concavuscutum and on fruits infested with both prey than on fruits with A. guerreronis only. The predators were attracted by odours emanating from coconuts with A. guerreronis, but not by odours from coconuts with S. concavuscutum, even when A. guerreronis were present on the same fruit. Fewer N. baraki were recaptured on fruits with both prey or with S. concavuscutum than on fruits with only A. guerreronis. Furthermore, the quality of A. guerreronis from singly and multiply infested coconuts as food for N. baraki did not differ. Concluding, our results suggest that N. baraki does not perform well when S. concavuscutum is present on the coconuts, and the control of A. guerreronis by N. baraki may be negatively affected by the presence of S. concavuscutum.

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

Coconut farming is not only a vital agricultural industry for all tropical countries possessing humid coasts and lowlands, but is also a robust income provider for millions of smallholder farmers worldwide. However, due to its longevity, the security of production of this crop suffers significantly from episodes of natural disasters, including cyclone and tsunami, devastating pest and disease outbreaks, while also affected by price competition for the principal products, especially the oil. In order to reduce these pressures, high-value coconut varieties (makapuno and aromatics) have been introduced in some regions, on a limited scale, but with positive outcomes. Even though these two varieties produce fruit with delicious solid or flavoursome liquid endosperm, their distinct biochemical and cellular features unfortunately prevent their in situ germination. In fact, embryo rescue and culture have been developed historically to nurture the embryo under in vitro conditions, enabling effective propagation. In an attempt to provide a comprehensive review featuring these elite coconut varieties, this paper firstly introduces their food values and nutritional qualities, and then discusses the present knowledge of their biology and genetics. Further possibilities for coconut in general are also highlighted, through the use of advanced tissue culture techniques and efficient seedling management for sustainable production of these highly distinct and commercially attractive varieties of coconut.

Estimated crop loss due to coconut mite and financial analysis of controlling the pest using the acaricide abamectin.

Reducing the losses caused by Aceria guerreronis Keifer has been an arduous task for farmers. However, there are no detailed studies on losses that simultaneously analyse correlated parameters, and very few studies that address the economic viability of chemical control, the main strategy for managing this pest. In this study the objectives were (1) to estimate the crop loss due to coconut mite and (2) to perform a financial analysis of acaricide application to control the pest. For this, the following parameters were evaluated: number and weight of fruits, liquid albumen volume, and market destination of plants with and without monthly abamectin spraying (three harvests). The costs involved in the chemical control of A. guerreronis were also quantified. Higher A. guerreronis incidence on plants resulted in a 60 % decrease in the mean number of fruits harvested per bunch and a 28 % decrease in liquid albumen volume. Mean fruit weight remained unaffected. The market destination of the harvested fruit was also affected by higher A. guerreronis incidence. Untreated plants, with higher A. guerreronis infestation intensity, produced a lower proportion of fruit intended for fresh market and higher proportions of non-marketable fruit and fruit intended for industrial processing. Despite the costs involved in controlling A. guerreronis, the difference between the profit from the treated site and the untreated site was 18,123.50 Brazilian Real; this value represents 69.1 % higher profit at the treated site.

De novo assembly and characterization of global transcriptome of coconut palm (Cocos nucifera L.) embryogenic calli using Illumina paired-end sequencing.

Production and supply of quality planting material is significant to coconut cultivation but is one of the major constraints in coconut productivity. Rapid multiplication of coconut through in vitro techniques, therefore, is of paramount importance. Although somatic embryogenesis in coconut is a promising technique that will allow for the mass production of high quality palms, coconut is highly recalcitrant to in vitro culture. In order to overcome the bottlenecks in coconut somatic embryogenesis and to develop a repeatable protocol, it is imperative to understand, identify, and characterize molecular events involved in coconut somatic embryogenesis pathway. Transcriptome analysis (RNA-Seq) of coconut embryogenic calli, derived from plumular explants of West Coast Tall cultivar, was undertaken on an Illumina HiSeq 2000 platform. After de novo transcriptome assembly and functional annotation, we have obtained 40,367 transcripts which showed significant BLASTx matches with similarity greater than 40 % and E value of ≤10(-5). Fourteen genes known to be involved in somatic embryogenesis were identified. Quantitative real-time PCR (qRT-PCR) analyses of these 14 genes were carried in six developmental stages. The result showed that CLV was upregulated in the initial stage of callogenesis. Transcripts GLP, GST, PKL, WUS, and WRKY were expressed more in somatic embryo stage. The expression of SERK, MAPK, AP2, SAUR, ECP, AGP, LEA, and ANT were higher in the embryogenic callus stage compared to initial culture and somatic embryo stages. This study provides the first insights into the gene expression patterns during somatic embryogenesis in coconut.

2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa.

Evidence of Amblyseius largoensis and Euseius alatus as biological control agent of Aceria guerreronis.

Amblyseius largoensis (Muma) (Acari: Phytoseiidae) and Euseius alatus De Leon (Acari: Phytoseiidae) are predatory mites that are mostly found on leaves and on the exposed fruit surface of coconut plants. Their morphology hampers the access to the microhabitat occupied by Aceria guerreronis Keifer (Acari: Eriophyidae), the most important pest of coconut fruits throughout the world. However, it was suggested that they can prey on A. guerreronis under natural conditions when this pest leaves its refuge to disperse. Since the trophic interactions between A. largoensis or E. alatus and A. guerreronis are unknown, we compare the frequencies of occurrence of A. largoensis and E. alatus under the bracts of coconut fruits and on coconut leaflets. In addition, because phytoseiids feed by liquid ingestion, we used molecular analysis to confirm the potential role of A. largoensis or E. alatus as predators of A. guerreronis and to assess how fast the A. guerreronis DNA fragment is degradated in the A. largoensis digestive tract. Our study demonstrated that E. alatus was only present on coconut leaflets whereas A. largoensis was found mostly on leaflets and, to a much lesser extent, under the bracts of coconuts. Species-specific ITS primers designed for A. guerreronis were shown to have a high degree of specificity for A. guerreronis DNA and did not produce any PCR product from DNA templates of the other insects and mites associated with the coconut agroecosystem. Based on molecular analysis, we confirmed that the predatory mites, A. largoensis and E. alatus, had preyed on the coconut mite in the field. Overall the predatory mites collected in the field exhibited low levels of predation (26.7% of A. largoensis and 8.9% of E. alatus tested positive for A. guerreronis DNA). The fragment of A. guerreronis DNA remained intact for a very short time (no more than 6 h after feeding) in the digestive tract of A. largoensis.

Coconut (Cocos nucifera l.) pollen cryopreservation.

BACKGROUND: Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. OBJECTIVE: The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. MATERIALS AND METHODS: Pollen of two coconut varieties (West Coast Tall WWCTW and Chowghat Orange Dwarf CODC) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. RESULTS: Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32 % in desiccated pollen whereas it was between 26 and 29 % in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31 % in desiccated pollen, while it was between 28 and 32 % in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 nm and 226-396 mm for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1 % after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively. CONCLUSION: These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

Whole genome sequencing and analysis of plant growth promoting bacteria isolated from the rhizosphere of plantation crops coconut, cocoa and arecanut.

Coconut, cocoa and arecanut are commercial plantation crops that play a vital role in the Indian economy while sustaining the livelihood of more than 10 million Indians. According to 2012 Food and Agricultural organization's report, India is the third largest producer of coconut and it dominates the production of arecanut worldwide. In this study, three Plant Growth Promoting Rhizobacteria (PGPR) from coconut (CPCRI-1), cocoa (CPCRI-2) and arecanut (CPCRI-3) characterized for the PGP activities have been sequenced. The draft genome sizes were 4.7 Mb (56% GC), 5.9 Mb (63.6% GC) and 5.1 Mb (54.8% GB) for CPCRI-1, CPCRI-2, CPCRI-3, respectively. These genomes encoded 4056 (CPCRI-1), 4637 (CPCRI-2) and 4286 (CPCRI-3) protein-coding genes. Phylogenetic analysis revealed that both CPCRI-1 and CPCRI-3 belonged to Enterobacteriaceae family, while, CPCRI-2 was a Pseudomonadaceae family member. Functional annotation of the genes predicted that all three bacteria encoded genes needed for mineral phosphate solubilization, siderophores, acetoin, butanediol, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, chitinase, phenazine, 4-hydroxybenzoate, trehalose and quorum sensing molecules supportive of the plant growth promoting traits observed in the course of their isolation and characterization. Additionally, in all the three CPCRI PGPRs, we identified genes involved in synthesis of hydrogen sulfide (H2S), which recently has been proposed to aid plant growth. The PGPRs also carried genes for central carbohydrate metabolism indicating that the bacteria can efficiently utilize the root exudates and other organic materials as energy source. Genes for production of peroxidases, catalases and superoxide dismutases that confer resistance to oxidative stresses in plants were identified. Besides these, genes for heat shock tolerance, cold shock tolerance and glycine-betaine production that enable bacteria to survive abiotic stress were also identified.

Identification and computational annotation of genes differentially expressed in pulp development of Cocos nucifera L. by suppression subtractive hybridization.

BACKGROUND: Coconut (Cocos nucifera L.) is one of the world's most versatile, economically important tropical crops. Little is known about the physiological and molecular basis of coconut pulp (endosperm) development and only a few coconut genes and gene product sequences are available in public databases. This study identified genes that were differentially expressed during development of coconut pulp and functionally annotated these identified genes using bioinformatics analysis. RESULTS: Pulp from three different coconut developmental stages was collected. Four suppression subtractive hybridization (SSH) libraries were constructed (forward and reverse libraries A and B between stages 1 and 2, and C and D between stages 2 and 3), and identified sequences were computationally annotated using Blast2GO software. A total of 1272 clones were obtained for analysis from four SSH libraries with 63% showing similarity to known proteins. Pairwise comparing of stage-specific gene ontology ids from libraries B-D, A-C, B-C and A-D showed that 32 genes were continuously upregulated and seven downregulated; 28 were transiently upregulated and 23 downregulated. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT), phospholipase D, acetyl-CoA carboxylase carboxyltransferase beta subunit, 3-hydroxyisobutyryl-CoA hydrolase-like and pyruvate dehydrogenase E1 ß subunit were associated with fatty acid biosynthesis or metabolism. Triose phosphate isomerase, cellulose synthase and glucan 1,3-ß-glucosidase were related to carbohydrate metabolism, and phosphoenolpyruvate carboxylase was related to both fatty acid and carbohydrate metabolism. Of 737 unigenes, 103 encoded enzymes were involved in fatty acid and carbohydrate biosynthesis and metabolism, and a number of transcription factors and other interesting genes with stage-specific expression were confirmed by real-time PCR, with validation of the SSH results as high as 66.6%. Based on determination of coconut endosperm fatty acids content by gas chromatography-mass spectrometry, a number of candidate genes in fatty acid anabolism were selected for further study. CONCLUSION: Functional annotation of genes differentially expressed in coconut pulp development helped determine the molecular basis of coconut endosperm development. The SSH method identified genes related to fatty acids, carbohydrate and secondary metabolites. The results will be important for understanding gene functions and regulatory networks in coconut fruit.

Composition, physicochemical properties and thermal inactivation kinetics of polyphenol oxidase and peroxidase from coconut (Cocos nucifera) water obtained from immature, mature and overly-mature coconut.

Composition, physicochemical properties and enzyme inactivation kinetics of coconut water were compared between immature (IMC), mature (MC) and overly-mature coconuts (OMC). Among the samples studied, pH, turbidity and mineral contents for OMC water was the highest, whereas water volume, titratable acidity, total soluble solids and total phenolics content for OMC water were the lowest. Maturity was found to affect sugar contents. Sucrose content was found to increase with maturity, and the reverse trend was observed for fructose and glucose. Enzyme activity assessment showed that polyphenol oxidase (PPO) in all samples was more heat resistant than peroxidase (POD). Compared to IMC and MC, PPO and POD from OMC water showed the lowest thermal resistance, with D83.3°C=243.9s (z=27.9°C), and D83.3°C=129.9s (z=19.5°C), respectively.

Multifarious beneficial traits and plant growth promoting potential of Serratia marcescens KiSII and Enterobacter sp. RNF 267 isolated from the rhizosphere of coconut palms (Cocos nucifera L.).

Two plant growth promoting bacteria designated as KiSII and RNF 267 isolated from the rhizosphere of coconut palms were identified as Serratia marcescens and Enterobacter sp. based on their phenotypic features, BIOLOG studies and 16S rRNA gene sequence analysis. Both bacteria exhibited phosphate solubilization, ammonification, and production of indole acetic acid, ß-1, 3 glucanase activities and 1-aminocyclopropane-1-carboxylate-deaminase activity. They could also tolerate a range of pH conditions, low temperature and salinity (NaCl). In addition, S. marcescens KiSII exhibited N- fixation potential, chitinase activity, siderophore production and antibiotics production. Seed bacterization with these bacteria increased the growth parameters of test plants such as paddy and cowpea over uninoculated control in green house assay. In coconut seedlings, significant increase in growth and nutrient uptake accompanied with higher populations of plant beneficial microorganisms in their rhizospheres were recorded on inoculation with both the PGPRs. The present study clearly revealed that PGPRs can aid in production of healthy and vigorous seedlings of coconut palm which are hardy perennial crops. They offer a scope to be developed into novel PGPR based bioinoculants for production of elite seedlings that can benefit the coconut farming community and the coconut based ecology.

Conservation of coconut (Cocos nucifera L.) germplasm at sub-zero temperature.

Protocols are proposed for the low (-20 degree C) and ultra-low (-80 degree C) temperature storage of coconut (Cocos nucifera L.) embryos. A tissue dehydration step prior to storage, and a rapid warming step upon recovery optimized the protocol. The thermal properties of water located within embryos were monitored using differential scanning calorimetry (DSC). In the most efficient version of the protocol, embryos were dehydrated under a sterile air flow in a dehydration solution containing glucose (3.33 M) and glycerol (15 percent) for 16 hours. This protocol decreased the embryo water content from 77 to 29 percent FW and at the same time reduced the amount of freezable water down to 0.03 percent. The dehydrated embryos could be stored for up to 3 weeks at -20 degree C (12 percent producing normal plants upon recovery) or 26 weeks at -80 degree C (28 percent producing normal plants upon recovery). These results indicate that it is possible to store coconut germplasm on a medium term basis using an ultra-deep freezer unit. However for more efficient, long term storage, cryopreservation remains the preferred option.

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification.

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

Differential responses to guano fertilization among tropical tree species with varying functional traits.

PREMISE OF THE STUDY: Seabirds often cause significant changes to soil properties, and seabird-dominated systems often host unique plant communities. This study experimentally (1) examined species-specific responses to seabird guano gradients, (2) considered the role that differential functional traits among species play in altering plant response to guano, and (3) investigated the implications of seabird guano on range-expanding species. METHODS: Using a greenhouse fertilization experiment, we examined how guano fertilization affects the growth and functional traits of four tree species dominant in the Pacific Islands: Cocos nucifera, Pisonia grandis, Scaevola sericea, and Tournefortia argentea. In these systems, seabirds are frequently found in association with three of these four species; the remaining species, C. nucifera, is a recently proliferating species commonly found in the region but rarely associated with seabirds. KEY RESULTS: We determined that responses to guano addition differed significantly between species in ways that were consistent with predictions based on differing functional traits among species. Notably, we demonstrated that C. nucifera showed no growth responses to guano additions, whereas all seabird-associated plants showed strong responses. CONCLUSIONS: These results provide experimental evidence of differential species response to guano additions, suggesting that differences in species functional traits may contribute to changes in plant communities in seabird-dominated areas, with seabird-associated species garnering performance advantages in these high-nutrient environments. Among these species, results also suggest that C. nucifera may have a competitive advantage in low-nutrient environments, providing an unusual example of how a range-expanding plant species can profit from low-nutrient environments.

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) acclimatization 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.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

GA3 stimulates the formation and germination of somatic embryos and the expression of a KNOTTED-like homeobox gene of Cocos nucifera (L.).

The micropropagation of coconut palm has progressed rapidly; yet, there are constraints with regard to the number of somatic embryos formed and their germination. To overcome these, we tested the effect of gibberellic acid and characterized genes of the KNOX family. Gibberellic acid at 0.5 muM increased 1.5-fold the number of calli forming somatic embryos and twofold the number of somatic embryos per callus, calli with germinating embryos and the number of germinating somatic embryos per callus. With regard to the study of KNOX family genes, the complete sequences of two KNOX-like genes were obtained for CnKNOX1 and CnKNOX2. The deduced amino acid sequence of both showed highly conserved domains characteristic of KNOX genes. CnKNOX1 showed high homology with KNOX class I proteins. CnKNOX1 expression was detected throughout the embryogenesis process except in somatic embryos at the pro-globular stage, and was highest in somatic embryos at the coleoptilar stage. No detection of CnKNOX1 expression occurred in calli with aberrant embryos. The addition of gibberellic acid stimulated the expression of CnKNOX1 earlier and the relative expression at all stages was higher. CnKNOX2 expression occurred at all stages peaking at the globular stage, but gibberellic acid treatment decreased the expression. Gene expression was also analyzed in tissues of different organs of adult palms. With CnKNOX1, high level of expression was found in tissues of organs with, but not in those without, meristem, whereas CnKNOX2 expression was detected in tissues with and also in those without meristem.

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

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.