Cryopreservation of Citrus limon (L.) Burm. F Shoot Tips
Cryopreservation of Citrus limon (L.) Burm. F Shoot Tips Using a Droplet-vitrification Method
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초록
This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: ‘FrostEureka limon’ and ‘Cook Eureka limon’, using a droplet-vitrification method. The shoot tips that were excised from in vitrogrown 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 loadingsolution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at 25℃. Priorto direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at 0℃ or PVS3at 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 werepost-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% forcryopreserved shoot tips of ‘Frost Eureka limon’ and ‘Cook Eureka limon’, respectively, when pre-cultured in 0.3 M and 0.5M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at 0℃. We also investigatedwhether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoottips. The viability of cooled samples, following culturing on woody plant media (WPM) containing ¼ ammonium nitrateovernight before micrografting, was the highest (70.3%) in ‘Frost Eureka limon’. The study described here is acost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruitplants and for breeding disease resistance.
Abstract
This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: ‘FrostEureka limon’ and ‘Cook Eureka limon’, using a droplet-vitrification method. The shoot tips that were excised from in vitrogrown 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 loadingsolution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at 25℃. Priorto direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at 0℃ or PVS3at 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 werepost-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% forcryopreserved shoot tips of ‘Frost Eureka limon’ and ‘Cook Eureka limon’, respectively, when pre-cultured in 0.3 M and 0.5M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at 0℃. We also investigatedwhether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoottips. The viability of cooled samples, following culturing on woody plant media (WPM) containing ¼ ammonium nitrateovernight before micrografting, was the highest (70.3%) in ‘Frost Eureka limon’. The study described here is acost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruitplants and for breeding disease resistance.
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