Cryopreservation of Arecanut (Areca catechu L.) Pollen.

BACKGROUND: Cryopreservation opens new avenues in the field of genetic resource conservation, especially in recalcitrant seeded palms such as arecanut for which field genebanks are exposed to pest and disease attacks and natural calamities. It is only through cryopreservation that the safety of the conserved germplasm can be assured at a relatively low cost for extended periods. OBJECTIVE: The objective of this work was to standardize various aspects of arecanut pollen cryopreservation, viz. collection and desiccation of pollen, in vitro germination, viability and fecundity studies. MATERIALS AND METHODS: Pollens of three arecanut genotypes (Sumangala, Hirehalli Dwarf and Hirehalli Dwarf x Sumangala) were collected in December 2013-February 2014. In vitro viability tests were conducted using fresh and desiccated pollen. Desiccated pollen was cryopreserved by direct immersion in liquid nitrogen and cryostored for different durations (24 hours to 2 years). Viability and fertility studies were conducted using cryopreserved pollen. RESULTS: Pollen extraction was achieved from fully opened male flowers by desiccation at room temperature (33-34 degree C). A medium containing 2.5 g/L sucrose was found to be best for in vitro germination at room temperature. There was no significant difference in germination between desiccated and cryopreserved pollen whereas pollen tube length decreased significantly after cryopreservation. Fertility studies using HD x Sumangala pollen cryostored for various durations (1 month, 1 year and 2 years) showed the setting of 70, 43 and 62%, respectively. Normal nut set was observed using cryopreserved pollen. CONCLUSION: Pollen cryopreservation is a viable option for germplasm conservation and hybridization programmes in arecanut.

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.

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.