Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

Barraco, Giuseppe,Sylvestre, Isabelle,Iapichino, Giovanni,Engelmann, Florent 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.2

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.

Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

Giuseppe Barraco,Isabelle Sylvestre,Giovanni Iapichino,Florent Engelmann 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.2

In this study, we investigated the possibility ofusing the droplet-vitrification technique for cryopreservingnodal segments of in vitro plantlets of the endangered plantspecies Lithodora rosmarinifolia. Among the three vitrificationsolutions tested, only solutions B1, containing (w/v)50 % glycerol and 50 % sucrose, and B3, containing 40 %glycerol and 40 % sucrose, were able to induce cryotolerancein nodal explants, resulting in intermediate survivaland recovery after cryopreservation. A three-step vitrificationprotocol, including an additional dehydration treatmentwith half-strength vitrification solution for 30 minbefore the treatment with full-strength vitrification solution,did not lead to any improvement in survival andrecovery compared with the two-step protocol. The optimalprotocol was the following: preculture of nodal segments inliquid medium with 0.3 M sucrose for 16 h and 0.7 Msucrose for 5 h, treatment for 20 min in loading solutioncontaining 1.9 M glycerol ? 0.5 M sucrose, dehydrationwith vitrification solution B1 (glycerol 50.0 %, sucrose50.0 %, w/v) for 60 min at room temperature, rapid coolingin minute droplets of vitrification solution, and rapidrewarming by immersion of nodal segments for 20 min inunloading solution containing 1.2 M sucrose. Under theseconditions, 33 % recovery of cryopreserved nodal explantswas achieved. Regrowth of cryopreserved samples wasrapid and direct. These results indicate that long-termstorage of L. rosmarinifolia by means of cryopreservationof nodal segments is possible, thereby contributing tosecuring the diversity of this rare and endangered plantspecies.

Cryopreservation of garlic germplasm collections using the droplet-vitrification technique.

Kim, Haeng-Hoon,Lee, Joung-Kwan,Hwang, Hae-Sung,Engelmann, Florent Royal Veterinary College 2007 Cryo letters Vol.28 No.6

<P>The droplet-vitrification protocol was applied to unripe inflorescences of plants of two Korean garlic collections, Danyang and Mokpo, to establish a cryopreserved germplasm collection. Garlic unripe inflorescences of the 59 accessions harvested at Danyang showed a mean survival of 83.3% and regeneration of 73.5% after cryopreservation. Unripe inflorescences of accessions cryopreserved at sub-optimal developmental stages displayed lower survival and/or regeneration. Of these 59 accessions, 53 were cryopreserved and stored for long-term conservation. In the Mokpo collection, unripe inflorescences of 149 accessions were cryopreserved, displaying a mean survival of 79.9% and regeneration of 78.2%. Of these 149 accessions, 116 were cryopreserved and stored for the long-term. A total of 252 accessions of five clonal Allium species, including garlic, were cryopreserved using unripe inflorescences, cloves or bulbils, with a mean survival of 80.9% survival and regeneration of 77.0%, from which 221 accessions were stored in liquid nitrogen for long-term conservation. The real-time quantitative, reverse transcription (RT)-PCR assay of several garlic viruses showed that virus concentration was much lower in plantlets originating from cryopreserved material, compared to plantlets originating from preculture control and dehydration control samples. These results demonstrate that large-scale implementation of cryopreservation of Allium germplasm is feasible and that it can result in the regeneration of virus-free or little infected material. These findings will strongly facilitate the conservation and international exchange of Allium germplasm.</P>

Thermal analysis of garlic shoot tips during a vitrification procedure.

Kim, Haeng-Hoon,Yoon, Ju-Won,Kim, Jung-Bong,Engelmann, Florent,Cho, Eun-Gi Royal Veterinary College 2005 Cryo letters Vol.26 No.1

<P>The thermal behavior of garlic shoot tips was analyzed during the course of a vitrification protocol using the PVS3 vitrification solution. The size of shoot tips did not significantly influence the thermal behavior of garlic shoot tips. Though there was no significance, endo-thermal enthalpy from melting of crystalline ice increased as preculture duration increased to 6 days. Preculture on medium with 0.5 M sucrose significantly lowered exo- and endothermal enthalpies of dehydration-control shoot tips. By contrast, after dehydration with PVS3 solution, the concentration of sucrose in preculture medium had no significant effect on the value of enthalpies. A big thermal event was observed in garlic shoot tips air-dried for 1-3 h before dehydration. Both vitrification solution and dehydration duration significantly (P < 0.0001) influenced exo- and endothermal enthalpies. After dehydration with PVS1, PVS2, Fahy or Steponkus solutions for 120 min, only a small peak was detected in some shoot tips, but recovery of cryopreserved shoot tips was low. Dehydration duration with PVS3 solution significantly (P < 0.0001) influenced exo- and endothermal enthalpies and onset temperatures during cooling and warming. After dehydration for 150 and 180 min with PVS3 vitrification solution, no crystallization was observed during cooling and warming in most replicates, and recovery of cryopreserved shoot tips was highest (> 80%). There was a significant (P < 0.001) negative correlation between moisture content of shoot tips and concentration of sucrose and glycerol, and regeneration of cryopreserved shoot tips. By contrast, there was a significant (P < 0.001) positive correlation between MC and enthalpy of ice melting, and onset temperature of crystallization. Overall, the results of the analysis of the thermal behavior of garlic shoot tips coincide very well with their recovery after cryopreservation and provide a very useful tool for the establishment and optimization of cryopreservation protocols.</P>

Cryopreservation of Cultivated and Wild Potato Varieties by Droplet Vitrification Procedure

Ju-Won Yoon,Haeng-Hoon Kim,Eun-Gi Cho,Ho-Cheol Ko,Hae-Sung Hwang,Young-Eun Park,Florent Engelmann 한국원예학회 2006 한국원예학회 기타간행물 Vol.- No.-

Implementation of Cryopreservation for Garlic Genetic Resources by the Droplet Vitrification Procedure

Haeng-Hoon Kim,Joung-Kwan Lee,Jae-Jun Ji,Sang-Sik Nam,Hae-Sung Hwang,Eun-Gi Cho,Florent Engelmann 한국원예학회 2006 한국원예학회 기타간행물 Vol.- No.-

Improved Cryopreservation Using Droplet-vitrification and Histological Changes Associated with Cryopreservation of Madder (Rubia akane Nakai)

Yi, Jung-Yoon,Sylvestre, Isabelle,Colin, Myriam,Salma, Mohammad,Lee, Sok-Young,Kim, Haeng-Hoon,Park, Hong-Jae,Engelmann, Florent Korean Society of Horticultural Science 2012 원예과학기술지 Vol.30 No.1

An efficient protocol for cryopreservation of madder hairy root cultures has been developed using droplet-vitrification. In previous study, combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective method. In this study, we tried three types of vitrification solution, B5, A3 (90% PVS2, on ice), and A5 (70% PVS2, on ice). Combining loading solution C4 and vitrification solution A5 (on ice) showed the best regeneration rate in this study. Histological changes of the cells within the hairy root of madder were also observed in different steps. The cells from the hairy roots of the control treatment were full and intact with different size of vacuoles and obvious cell nucleus having a dark nucleolus. After the stage of preparing for cryopreservation (after preculturing, loading, followed by dehydration solution A5 or B5), intercellular spaces had become distinct, and within cells, the cytoplasms had become denser and week plasmolyses had appeared. The cell plasmolyses were much more apparent and we measured the degree of plasmolysis by calculating, the area of cell/the area of cytoplasm. The value of plasmolysis degree was the highest in the combination of preculture, loading solution C4, and dehydration solution A5, 1.97. Because the highest regeneration rates appeared in the treatment of A5 for 20 min, we could assume that the optimal degree of plasmolysis for cryopreservation might be around 1.97. The changes in cell structure during cryopreservation might be a useful basis for the development of a proper long-term preservation method for madder germplasms.

Cryopreservation of Citrus madurensis Zygotic Embryos by Modified Encapsulation-dehydration

Eun Gi Cho,Yue Luan Hor,Haeng Hoon Kim,Jae Gyun Gwag,V. Ramanatha Rao,Florent Engelmann 한국육종학회 2003 한국육종학회지 Vol.35 No.3

The rol e of osm otol erance and dehydr ation, in terms of both medium com posi tion and exposur e duration, on sur vi val ofem bryos of Citrus m adurensi s after cryopr eser vation usi ng the encapsul ation-dehydr ation and modified encapsul ation-dehydr

Changes in sucrose and glycerol content in garlic shoot tips during freezing using PVS3 solution.

Kim, Jung-Bong,Kim, Haeng-Hoon,Baek, Hyung-Jin,Cho, Eun-Gi,Kim, Yong-Hwan,Engelmann, Florent Royal Veterinary College 2005 Cryo letters Vol.26 No.2

<P>Changes in moisture content (MC), sucrose and glycerol concentration in garlic shoot tips were monitored during loading and unloading with PVS3 solution. Upon PVS3 treatment, shoot tip MC decreased rapidly and sucrose and glycerol concentrations increased rapidly during the first 30 min. Sucrose and glycerol concentrations increased more slowly thereafter. Shoot tip MC in after PVS3 treatment was affected by their size, but not by sucrose concentration of the preculture medium. As the size of shoot tips increased, so their MC increased after PVS3 treatment. However, sucrose and glycerol concentrations decreased after PVS3 incubation, and concentrations in dehydrated shoot tips were much lower than those measured in non-air dried controls. During unloading with 1.2 M sucrose medium, shoot tip MC increased rapidly during the first 10 min, whereas glycerol concentration decreased steadily over 90 min. Loading and unloading of PVS3 solution in garlic shoot tips follows the principle of solute bulk flow.</P>

Improved Cryopreservation Using Droplet-vitrification and Histological Changes Associated with Cryopreservation of Madder (Rubia akane Nakai)

Jung-Yoon Yi,Isabelle Sylvestre,Myriam Colin,Mohammad Salma,Sok-Young Lee,Haeng-Hoon Kim,Hong-Jae Park,Florent Engelmann 한국원예학회 2012 원예과학기술지 Vol.30 No.1

An efficient protocol for cryopreservation of madder hairy root cultures has been developed using droplet-vitrification. In previous study, combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective method. In this study, we tried three types of vitrification solution, B5, A3 (90% PVS2, on ice), and A5 (70% PVS2, on ice). Combining loading solution C4 and vitrification solution A5 (on ice) showed the best regeneration rate in this study. Histological changes of the cells within the hairy root of madder were also observed in different steps. The cells from the hairy roots of the control treatment were full and intact with different size of vacuoles and obvious cell nucleus having a dark nucleolus. After the stage of preparing for cryopreservation (after preculturing, loading, followed by dehydration solution A5 or B5), intercellular spaces had become distinct, and within cells, the cytoplasms had become denser and week plasmolyses had appeared. The cell plasmolyses were much more apparent and we measured the degree of plasmolysis by calculating, the area of cell/the area of cytoplasm. The value of plasmolysis degree was the highest in the combination of preculture, loading solution C4, and dehydration solution A5, 1.97. Because the highest regeneration rates appeared in the treatment of A5 for 20 min, we could assume that the optimal degree of plasmolysis for cryopreservation might be around 1.97. The changes in cell structure during cryopreservation might be a useful basis for the development of a proper long-term preservation method for madder germplasms.