Korean Journal of Plant Resources (한국자원식물학회지)

The Plant Resources Society of Korea (한국자원식물학회)
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A Successful Regeneration from Shoot Tips of Chrysanthemum morifolium (Ramat.) following Cryopreservation by Droplet-vitrification

  • Received : 2018.11.12
  • Accepted : 2018.12.23
  • Published : 2018.12.31
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Abstract

This study describes an efficient and widely applicable droplet-vitrification following cryopreservation for shoot tips of Chrysanthemum morifolium (Ramat.) cvs. 'Borami' and 'Yes morning'. The shoot tips of Chrysanthemum were precultured in Murashige and Skoog (MS) liquid medium supplemented with sucrose (0.3-0.7 M). Precultured explants were treated with loading solution (LS, C6) containing glycerol 20% and sucrose 20% for 30 min and exposed to dehydration solution (B5) containing 40% of glycerol and 40% of sucrose for 60 min at $25^{\circ}C$, and then transferred onto droplets containing $2.5{\mu}l$ PVS3 on sterilized aluminum foils ($4cm{\times}0.5cm$) prior to direct immersion in liquid nitrogen (LN) for 1 h. The highest regeneration rate (%) was obtained when shoot tips were precultured with treatment-2 (exposing of shoot tips to MS + 0.3M sucrose for 30 h and then treated with MS+0.5 M sucrose for 16 h) at $25^{\circ}C$ in both the cultivars. The viability of cooled samples, followed by culturing on $NH_4NO_3$-free MS medium for first 5 days was increased to two-fold (80.7%) regrowth rate over those cultured on normal MS medium or MS medium containing plant growth regulators. This result shows droplet-vitrification would be a promising method for cryobanking chrysanthemum germplasm.

Keywords

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Fig. 1. Schematic diagram showing the process of cryopreservation of shoot tips of C. morifolium by droplet-vitrification and regeneration under in vitro conditions.

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Fig. 2. Cryopreservation of shoot tips of C. morifolium L. cv. ‘Borami’ by droplet-vitrification. (a) In vitro grown plantlets of Chrysanthemum used for cryopreservation experiments. (b) Nodal explants cultured on MS medium for the emergence of shoot tips. Enlarged form of nodal explant (indicated by arrow). (c) Appearance of shoot tip from nodal axially bud (shoot tip is rounded by circle). (d) dissected shoot tip used for cryopreservation. (e) Dehydration of shoot tips in vitrification solution (B5) droplets on sterilized aluminum foil strips. (f) Immersion of aluminum foil along with shoot tips into the liquid nitrogen (LN) for 1 h. (g) ) Plant regeneration from cryopreserved shoot tips 8 weeks after inoculation. (h) A shoot regenerated into whole plant from cryopreserved shoot tips that had been cultured on post-culture medium (PCM-2) containing NH4NO3-free MS medium. (i) Acclimatized plantlet from cryopreserved (+LN) shoot tips (right), and treated control (-LN) shoot tips (left) under greenhouse conditions. c, d, e) Bar = 0.5 ㎝ ; a, b, f, g, h, i) Bar = 1.0 ㎝.

Table 1. Effect of preculture treatment on regeneration rates of chrysanthemum shoot tips before (-LN) and after cryopreservation (+LN) following liquid nitrogen immersionz

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Table 2. Effect of loading and dehydration solutions and exposure time on the regeneration rate of chrysanthemum shoot tips before (−LN) and after liquid nitrogen exposure (+LN)

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Table 3. Effects of post-culture media on regeneration rate (%) of the treated control (−LN) and cryopreserved (+LN) shoot tips of the two cultivars of C. morifolium by droplet-vitrification

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