Cryopreservation of coconut plumule using droplet vitrification

In the present investigation, four types of explants from mature zygotic embryos of coconut, viz., whole upper cotyledonary region without haustorium, half of the upper cotyledonary region without haustorium, plumule with a portion of radicle and exclusively plumular tissue, were cultured in 12 different media combinations to find a suitable explant which could be regenerated after cryopreservation. Explants were pre-cultured in medium with 0.4 and 0.5 M sucrose for three days followed by dehydration in PVS3 solution for different durations on a sterile aluminum strip after treating with loading solution. Strips were treated with liquid nitrogen inside a cryoflask until bubbling stopped and quickly transferred to a cryovial and stored for a minimum period of 24 hours in liquid nitrogen. It was observed that plumule alone or with a small portion of outer tissue was ideal for fast in vitro growth and recovery of whole plantlets of coconut in a medium supplemented with NAA alone. Addition of glutamine (5 mg L-1), TDZ (1 mg L-1) and NAA (18 mg L-1) aided the vigorous growth of plantlets. In control, the survival rate ranged from 60 to 90 per cent in plumule pre-grown in media containing 0.5 M sucrose after dehydration with PVS3 for various durations, whereas it was 14 to 75 per cent in cryopreserved ones. Considering the high survival (75%) and regrowth (35%) of cryopreserved plumule in the present study, there is much scope for further improvement of the procedure to find the right combination of factors so as to enhance complete recovery of plantlets without much injury to plumules during cooling and rewarming

Histological studies of cellular differentiation during somatic embryogenesis of coconut plumule-derived calli

Since coconut is   one of the most recalcitrant species to generate in vitro, it is   necessary to study in detail about the cellular changes that occur during   somatic embryogenesis to enhance our knowledge about this phenomenon. In the   present study, coconut plumular tissues, the shoot meristem including leaf   primordia, were used as explants for in vitro regeneration studies.   Histological studies were carried out in different stages of plumule culture.   No noticeable growth was observed in 15 days old cultures. After 30 days,   meristematic cells could be identified. Abundance of meristematic cells,   foremost to the development of callus structures, was observed after 45 days.   After 75 days, globular friable calli were formed and histological studies   revealed the presence of meristematic centers which eventually formed somatic   embryos. The histological study of matured somatic embryos formed after 120   days of callus initiation showed a clear meristematic zone of parenchyma   cells, surrounded by vascular bundles. Histological studies, carried out for   certain abnormalities like compact calli, abnormal somatic embryoids with   rudimentary shoots and multiplied roots, revealed the presence of intact   cotyledonary leaves which seemed to inhibit the apical meristem development   of somatic embryoids. The presence of vascular bundles in the early stages of   callus formation might lead to the direct formation of meristemoids. These   results could aid future studies leading to enhanced control of the somatic   embryogenic process and greater efficiency of somatic embryo and plantlet   formation in coconut

Estimation of out-crossing rates in populations of West Coast Tall cultivar of coconut using microsatellite markers

Understanding of mating system of a plant species has fundamental importance for formulation of genetic conservation strategies and breeding programmes. The pattern of gene flow, via pollen, has a profound influence on the genetic structure within a population. Various genetic parameters, obtained from molecular marker studies, can be used to assess estimates of mating system. The aim of this study was to estimate the rate of outcrossing in West Coast Tall (WCT) cultivar of coconut, which is predominant in India, using microsatellite simple sequence repeats (SSR). Two WCT mother palms and their 88 progenies, collected as embryos for five months, were screened using 15 highly polymorphic microsatellite primers. The mating parameters were estimated using mixed mating model (MLTR software) and the extents of similarity between the mother palms and their progenies were analyzed using the NTSYS software. The percentage similarity between the mother palm and its progenies, as deduced using microsatellite data, ranged from 55 to 74 per cent. The progenies were also analyzed using a RAPD primer capable of distinguishing Tall and Dwarf palms. All the progenies were found to possess the Tall-type marker indicating that the pollen was derived from Tall palms in all the cases. The results revealed the WCT cultivar to be pre-dominantly out-crossing and indicated that proper sampling and indicated that proper sampling and breeding strategies are required to sustain the high genetic diversity found

Maintenance of embryogenic potential of calli derived from embryonic shoot of West Coast Tall cv. of coconut (Cocos nucifera L.)

Maintenance of embryogenic potential of calli is important as the totipotency is often lost in a short time in vitro. This caters to the need for year round availability of somatic embryos in a regenerable state. In the present study, 14 media combinations, with either 2,4-D or picloram as auxin source, were tested for maintaining embryogenic calli obtained from embryonic shoot explants of coconut. Irrespective of type and concentration of auxins, callusing was observed in all the media combinations. However, high dose of 2,4-D (above 74.6 μM) in the initial medium resulted in intense browning and lesser percentage of callusing. Embryogenic nature of calli could be maintained to a maximum of 21 weeks in medium supplemented with 2,4-D (74.6 μM) and subsequent culturing into higher concentration of 2,4-D (90.4 μM). Gene expression studies carried out using qRT-PCR revealed that genes such as ECP, GST, LEAFY and WUS were highly expressed in long term embryogenic calli (21 week old) and genes such as SERK, GLP, WRKY and PKL in initial embryogenic calli (21 days old). The study concludes that coconut plumular calli could be maintained for longer periods without compromising on the embryogenic potential of the calli

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Not AvailableAn attempt was made to establish highly competent embryogenic cell suspension culture in coconut, a species recalcitrant to in vitro culture. Embryogenic calli were initiated from shoot meristem explants of coconut. Y3 medium supplemented with 2.4-D (4.5 μM) and glutamine (34.2 μM) was found to be the best medium to initiate cell suspension. Growth evaluation was done by packed cell volume (PCV) and it was found that maximum growth volume of 9.9% was reached at 200 days of culture initiation. About 52% of viable cells were detected through fluorescent microscopy. Cell aggregation was noticed in Y3 medium supplemented with glutamine (34.2 μM) , malt extract (100mg/l), biotin (40.9 μM) and kinetin (9.3 μM), but further progress could not be achieved. It was also observed that embryogenic calli were not of a friable type, but were associated with densely aggregated cells. Because of its hard nature, we were unsuccessful to obtain high quality cell suspension.Not Availabl

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Not AvailableBACKGROUND: 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°C), A medium containing 2,5 giL 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 (l 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.Not Availabl

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