Cryopreservation of Citrus limon (L.) Burm. F Shoot Tips Using a Droplet-vitrification Method

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 $25^{\circ}C$ 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 $25^{\circ}C$. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at $0^{\circ}C$ or PVS3 at $25^{\circ}C$. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in $\text\tiny{^1/_2}$ MS for 30 min at $25^{\circ}C$. 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 $0^{\circ}C$. 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 $\text\tiny{^1/_4}$ 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.

Fig. 1. Regrowth from cryopreserved shoot tips of Citrus limon by droplet-vitrification after micrografting. (a) Etiolated ‘trifoliate orange’ seedling rootstocks were prepared by making notched incisions 1.0 ㎝ above the cotyledonary node under a stereo microscope. (b) A typical dissected shoot tip in an enlarged form. (c) Citrus shoot tips contained in droplets of vitrification solution on aluminum foil strips (40 × 5 ㎜). (d) Trimmed shoot tips after post-thaw culture were placed on the notch of the seedling rootstock. (e) Survival of a micrografted shoot tip on the rootstock seedlings of ‘trifoliate orange’. (f) Enlarged form of micrografted shoot tip on the rootstock after 15 days. (g) Recovery of micrografted shoot tips on etiolated ‘trifoliate orange’ seedling rootstocks for 8 weeks. (h) Comparison of non-treated control (con), no liquid nitrogen (LN) exposure (−LN) and LN exposure (+LN) for 6 weeks after micrografting. Ten shoot tips of each cultivar were used for the experiment and each experiment was repeated twice. a, b, d, f: scale bar = 0.5 ㎝; c, e, g, h: scale bar = 1.0 ㎝.

Fig. 2. Effects of various preculture treatments on the regeneration rates (%) of treated control (−LN) and cryopreserved (+LN) shoot tips of two cultivars: ‘Frost Eureca limon’ (a) and ‘Cook Eureca limon’ (b) of Citurs limon. Shoot tips were pre-cultured on Murashige and Skoog (MS) medium that contained sucrose at 0.3 M and 0.5 M concentrations and incubated for different durations. The various pre-culture media and time durations were as follows: MS + 0.3 M Sucrose for 24 h (Medium 1); MS + 0.3 M Sucrose for 24 h and then treated in MS + 0.5 M sucrose for 16 h (Medium 2); MS + 0.3 M Sucrose for 48 h (Medium 3); MS + 0.3 M sucrose for 48 h and then treated in MS + 0.5 M sucrose for 16 h (Medium 4); MS + 0.3 M Sucrose for 72 h (Medium 5); MS + 0.3 M Sucrose for 72 h and then treated in MS + 0.5 M sucrose for 16 h (Medium 6); followed by loaded and dehydrated with PVS2 at 0℃ or PVS3 at 25℃, prior to direct immersion in LN for 1 h. The results are presented as means ± SE. The same lowercase letters indicate that the bar values do not differ significantly between each other according to LSD (P = 0.05).

Fig. 3. Effects of PVS2 and PVS3 exposure on regrowth of the treated control (−LN) and cryopreserved (+LN) Citrus shoot tips in two cultivars cooled to -196℃. Effects of PVS2 and PVS3 duration on regrowth rate (%) of treated control (−LN) and cryopreserved (+LN) shoot tips of citrus cultivars: ‘Frost Eureca limon’ (A & B) and ‘Cook Eureca limon’ (C & D). Pre-cultured shoot tips (MS + 0.3 M Sucrose for 48 h and then treated with MS + 0.5 M sucrose for 16 h) were loaded and dehydrated with PVS2 or PVS3 at 0℃ or 25℃, respectively, prior to direct immersion in LN for 1 h. Regeneration was recorded after 30 days of post-culture. The results are presented as means ± SE. The same lowercase letters indicate that the bar values do not differ significantly between each other according to LSD (P = 0.05). PVS2: 30% glycerol + 15% DMSO + 15% ethylene glycol + 13.7% sucrose in MS; PVS3: 50% glycerol + 50% sucrose in MS.

Fig. 4. Effects of different post-culture media on the recovery of shoot tips of two cultivars ‘Frost Eureca limon’ (A) and ‘Cook Eureca limon’ (B) of Citrus limon after LN exposure. Shoot tips were pre-cultured for 2 d at 24℃ on media with 0.3 M sucrose for 48 h and 0.5 M sucrose for 16 h, followed by a loading solution and dehydration with PVS2 at 0℃ or PVS3 at 25℃, prior to direct immersion in LN for 1 h. Shoot tips were rewarmed at 40℃, unloaded for 20 min in a medium with 0.5 M sucrose, and transferred to Murashige and Skoog (MS) media or woody plant media (WPM) with the following combinations: MS medium containing NH₄NO₃ (post-culture medium-1; PCM-1), MS medium without NH₄NO₃ (post-culture medium-2; PCM-2), woody plant medium (WPM) containing ¼ NH₄NO₃ (post-culture medium-3; PCM-3), WPM containing ½ NH4NO3 (post-culture medium-4; PCM-4), pH of the media was adjusted to 5.8 before adding 0.05% activated charcoal to the medium. The shoot tips un-treated with LN (− LN) served as control. The regeneration rate (%) of the cryopreserved shoot tips was recorded 8 weeks after inoculation. The results are presented as means ± SE. The same lowercase letters indicate the bar values do not differ significantly between each other according to LSD (P = 0.05).