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2018 Vol.31, Issue 6 Preview Page

Research Article

Cryopreservation of Citrus limon (L.) Burm. F Shoot Tips Using a Droplet-vitrification Method
Jung-Yoon Yi1
,
Kotnala Balaraju2
,
Hyung-Jin Baek3
,
Mun-Seop Yoon3
,
Haeng-Hoon Kim4
,
Young-Yi Lee1*
1Researcher, National Agrobiodiversity Center, NAS, RDA
2Post-doc, National Agrobiodiversity Center, NAS, RDA
3Senior Researcher, National Agrobiodiversity Center, NAS, RDA
3Professor, Department of Well-being Resources, Sunchon National University
*Corresponding Author
31 December 2018. pp. 684-694
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Abstract

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: ‘Frost Eureka limon’ and ‘Cook Eureka limon’, using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at 25℃ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at 25℃. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at 0℃ or PVS3 at 25℃. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in ½ MS for 30 min at 25℃. Shoot tips were post-cultured overnight on survival medium and then micrografted onto ‘trifoliate orange’ (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of ‘Frost Eureka limon’ and ‘Cook Eureka limon’, respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at 0℃. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing ¼ ammonium nitrate overnight before micrografting, was the highest (70.3%) in ‘Frost Eureka limon’. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

Keywords
Citrus
Cryopreservation
Droplet-vitrification
Micrografting
Shoot tip
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Information
  • Publisher :The Plant Resources Society of Korea
  • Publisher(Ko) :한국자원식물학회
  • Journal Title :Korean Journal of Plant Resources
  • Journal Title(Ko) :한국자원식물학회지
  • Volume : 31
  • No :6
  • Pages :684-694
  • Received Date : 2018-11-28
  • Revised Date : 2018-12-24
  • Accepted Date : 2018-12-27
  • DOI :https://doi.org/10.7732/kjpr.2018.31.6.684
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