PVS2 Treatment Research Articles

Cryoconservation of In Vitro Grown Shoot Tips of Cicer Microphyllum: A Crop Wild Relative of Chickpea

BACKGROUND: Cicer microphyllum Benth. is a crop wild relative (CWR) of chickpea (C. arietinum L.), that possess useful genes for cold and drought tolerance. The species is being conserved in the In Vitro Active Genebank for short- to medium – term conservation. Cryopreservation would be a useful complementary approach for its long-term conservation. OBJECTIVE: The current work aimed to develop an efficient cryoconservation protocol for cryobanking of C. microphyllum shoot tips. MATERIALS AND METHODS: In vitro shoot tips excised from 4-month old shoot cultures grown on B5 + 0.5 mg L-1 KIN + 0.1 mg L -1 NAA + 10 mg L -1 AgNO 3medium were cryoconserved using a droplet-vitrification technique. Post-thaw regrowth was evaluated after: (i) preculture medium (B5 basal, B5 + 3, 4, 6 and 10% sucrose), (ii) preculture incubation temperature (25 ± 2, 10, 8 and 22/5°C), (iii) PVS2 duration (10, 20, 30. 40, 50 and 60 min) and (iv) regrowth medium (B5) supplemented with 0.5 mg L-1 KIN + 0.1 NAA mg L-1 ; 0.5 mg L-1 KIN + 0.1 mg L-1 NAA + 10 mg L-1 AgNO3; 0.2 mg L-1 BAP + 10 mg L-1 AgNO3 ; 0.2 mg L-1 BAP + 20 mg L-1 AgNO3 and 0.2 mg L-1 BAP + 30 mg L-1 AgNO3,. RESULTS: In vitro shoot tips grown on B5 + 0.5 mg L-1 KIN + 0.1 mg L-1 NAA + 10 mg L-1 AgNO3, precultured on B5 + 6% sucrose at 10°C for 3 days, followed by PVS2 treatment for 20 min, unloading solution for 60 min and regrowth on B5 + 0.2 mg L-1 BAP + 20 mg L-1 AgNO3 resulted in highest survival (57%) and regrowth (40%) after cryoconservation. CONCLUSION: The standardized protocol was successfully used for cryobanking of in vitro shoot tips of C. microphyllum in the In Vitro Base Genebank of ICAR-NBPGR, New Delhi.

In Vitro Multiplication Rate and Pot Yield of Cryopreservation Derived Buds of Yams (Dioscorea Spp)

One of the principal objectives of the germplasm preservation is to maintain their unique genes combinations. A study was conducted in the laboratory and glass house to evaluate the multiplication rate of in vitro cultured plantlets, several morphological and yield characters of glass house pots grown plants derived from cryopreservation using modified droplet method on yams (Dioscorea spp.). In vitro MR was similar among in vitro culture, PVS2 treatment and cryopreservation derived plantlets for D. polystachya and D. cayenensis. In vitro only derived plantlets exhibited significantly lower MR in comparison to the two other treatment variants for D. bulbifera. This was, however, could be attributed to physiological effects which will be recovered in further sub-culture cycles. The morphological characters, number of tuber per plant and tuber' harvest weight per plant were constant for plant derived after cryopreservation and in vitro culture (control) for D. cayenensis. Modified droplet method is, therefore, reliable for the cryopreservation of yams germplasm and maybe other crops.

Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources

Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3–9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.

Cryopreservation of grapevine (Vitis spp.) shoot tips from growth chamber-sourced plants and histological observations

Many genebanks rely on cryopreservation as a method to preserve vulnerable field collections of vegetatively propagated crops. Effective cryopreservation procedures have been identified for Vitis; however, they usually use in vitro plantlets as the shoot tip source materials. It is costly to establish Vitis collections in vitro prior to cryopreservation. We sought to determine if growth chamber derived Vitis plants could serve as the source of shoot tips for cryopreservation. Nodal sections from growth chamber derived plants were surface-disinfected and placed in tissue culture on pre-treatment medium for 2 weeks. Uniform apical shoot tips (1 mm) were first obtained from the nodal sections and then precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), ascorbic acid and plant preservative mixture. Half-strength PVS2 was applied for 30 min at 22 °C, prior to full-strength PVS2 treatment at 0 °C. Cryopreserved shoot tips had the highest average regrowth of 50 and 55 % without and with cold-acclimation followed with a full-strength PVS2 exposure duration of 40 and 30 min at 0 °C, respectively. This cryopreservation protocol achieved high percentages of regrowth in V. vinifera 'Chardonnay' and 'Riesling' and V. hybrid 'Oppenheim'. Histological observations revealed that shoot tips from growth chamber plants had apical as well as multiple lateral meristems that survived LN immersion. The preservation of multiple meristems in each shoot tip may increase the capacity of shoot tip regeneration in cryopreserved Vitis that originates from ex vitro sources. The high percentage of regrowth after shoot tip cryopreservation using Vitis shoot tips derived from growth chamber source plants suggest that it may be possible to cryopreserve Vitis shoot tips without first introducing each accession into tissue culture.

Cryopreservation of Habenaria radiata and Habenaria rhodocheila seeds using the aluminum cryo-plate vitrification method

The genus Habenaria consists of 600 species of terrestrial orchids distributed in Asia, America and Africa. Habenaria rhodocheila Hance is found in Thailand, Myanmar, Cambodia and Laos. Habenaria radiata (Thunb.) K. Spreng is native to Japan. The number of both species has decreased in their habitats because of overcollecting and environmental changes in the habitats. Conservation of the surviving plants is an urgent issue to maintain the genetic diversity of the species. Seeds of H. radiata and H. rhodocheila were surface sterilized, desiccated in a laminar air-flow cabinet, and then subjected to the cryo-plate vitrification method. Viability of the seeds with or without immersion in liquid nitrogen (LN) was estimated by seed germination. The seeds of H. radiata showed high viability after immersion in LN without PVS2 treatment, and the viability decreased when exposure to PVS2 exceeded 40 min. On the other hand, viability of the H. rhodocheila seeds after immersion in LN increased with PVS2 treatment for 10 to 30 min, stayed at high levels with 40 to 60 min of PVS2 treatment, and then decreased with more than 90 min of PVS2 treatment.

Cryopreservation of Hamilin sweet orange [(Citrus sinensis (L.) Osbeck)] embryogenic calli using a modified aluminum cryo-plate technique

A modified aluminum cryo-plate technique was applied for cryopreservation of embryogenic citrus calli using aluminum cryo-plate. After encapsulation in a medium with 3% alginate, the beads were placed directly on aluminum foil troughs for exposure to plant vitrification solution (PVS2) incubated on ice for three intervals (30, 45 and 60min). After rewarming, the calli were incubated under light (16h/8h) or dark at 27±2°C. The survival rates ranged from 59.2% following 45min of PVS2 exposure to 88.7% after 30min of exposure. The 30-min PVS2 treatment yielded the highest callus growth (1.4) and percentage of calli with embryos regenerated (43.57%). The highest percentage of embryos regenerated was observed in dark condition (75.2%). The method described using aluminum foil troughs is efficient for cryopreservation of embryogenic citrus calli.

Integration of Cryopreservation and Tissue Culture for Germplasm Conservation and Propagation of Rosa pomifera ‘Karpatia’

Cryopreservation is an useful technique for long-term conservation that requires minimal space and maintenance. Germplasm protection of Rosa is important to preserve genetic diversity, to store material for breeding and to expand new research. This study was conducted to develop a droplet vitrification cryopreservation and micropropagation of Rosa pomifera cv. ‘Karpatia’, whose large hypanthia are characterized by remarkable pro-health properties. Culture in vitro was stabilized and shoot tips collected from dormant buds served as initial explants. The multiplication of shoots was carried out on MS medium containing benzyladenine. For the droplet vitrification cryopreservation, shoot tips from in vitro cultures were used: small with exposed meristem, and large with a meristem covered with leaves, as well as shoot tips from in situ plants, which were collected in winter. Treatment time with plant vitrification solution (PVS2) was also tested (10-30 minutes). From in vitro culture, 32-41% small explants with exposed meristem survived, but they regenerated at a very low level. The best cryostorage results were obtained for shoot tips from dormant buds and a 20-minute PVS2 treatment: the survival was 84% and regeneration 72%. During the post-freezing regeneration multiplication index was 2.4 shoots per one multiplication cycle, after cryopreservation and in the control. On half MS medium without growth regulators, 97-99% of shoots rooted, and all rooted plants have adapted to ex vitro conditions and were planted into the soil. Biometric analyses during shoot multiplication, rooting and acclimatization stages did not reveal any changes compared to the non-cryopreserved samples.

Storage behavior and cryopreservation studies in Indian rough lemon (Citrus jambhiri): a promising rootstock for long-term conservation

The study of the seed storage behavior and cryopreservation of embryonic axes was attempted in Citrus jambhiri genotypes using air desiccation-freezing, vitrification, and encapsulation-dehydration methods. Seeds of this species revealed nonorthodox seed storage behavior with moderate seed longevity and sensitivity to desiccation and freezing. Successful cryopreservation was achieved with all three methods using embryonic axes; however, the recovery growth percentage significantly differed among these methods. Cryopreservation was efficient for the embryonic axes desiccated to 13%-15% moisture content. Freezing of embryonic axes at this moisture content in liquid nitrogen showed a good recovery rate of 40%-50%. In the vitrification method, embryonic axes subjected to PVS2 treatment for 40 min had the highest recovery rate of 25% after cryopreservation. In the encapsulation-dehydration method, encapsulated axes precultured on 0.5 M sucrose medium followed by 6 h of desiccation had the highest recovery growth of 30%-60% after cryopreservation. Out of the three methods employed, the encapsulation-dehydration method gave the best recovery values for embryonic axes. High regeneration values of embryos in comparison to embryonic axes prompted us to undertake large-scale cryobanking of C. jambhiri using embryos (seeds without seed coats) and more than 100 accessions of C. jambhiri have been cryostored in the national cryobank. This would further serve the purpose of conserving nucellar embryos with zygotic embryos.

Comparison of droplet-vitrification and D-cryoplate for cryopreservation of date palm (Phoenix dactylifera L.) polyembryonic masses

In this work we tested the efficiency of two techniques, droplet-vitrification (DV) and dehydration (D) cryo-plate, for cryopreservation of polyembryonic masses (PEMs) of two date palm varieties, Sokary and Sultany. With DV, recovery of non-precultured, cryopreserved PEMs was nil without treatment with the PVS2 vitrification solution, which contained 3.3M glycerol+2.4M ethylene glycol+0.4M sucrose+1.9M dimethylsulfoxide. Following PVS2 treatments between 15 and 120min, it was comprised between 90.9–98.6% and 85.6–88.0% for varieties Sokary and Sultany, respectively. Sucrose preculture (3 days, 0.5M) led to 21.1% recovery of cryopreserved PEMs of variety Sokary only without PVS2 treatment, and slightly reduced recovery in all other experimental conditions with both varieties, compared with non-cryopreserved PEMs. Regrowth intensity of cryopreserved PEMs was generally lower with variety Sultany compared to variety Sokary. With the D cryo-plate technique, no recovery of cryopreserved PEMs was achieved without sucrose preculture. Sucrose preculture (3 days, 0.5M) had a positive effect on recovery of cryopreserved PEMs. For variety Sokary, the highest recovery (92.0–95.8%) was achieved for desiccation periods between 60 and 120min. Recovery was between 67.0 and 74.6% after desiccation periods of 90–120min for variety Sultany. With the D cryo-plate technique, regrowth intensity of cryopreserved PEMs was higher with variety Sokary compared to variety Sultany.

Anatomy and osmotic potential of the Vitis rootstock shoot tips recalcitrant to cryopreservation

This study was carried out on Kober 5BB (Vitis Berlandieri × V. riparia) grape rootstock shoot tips during the preparatory steps preceding the direct immersion in liquid nitrogen, in order to overcome until now unsuccessful cryopreservation with this species. The exposure of shoot tips to 0.3-0.4 M sucrose leads to a high cell solute concentration. The treatment with plant vitrification solution (PVS2) alone, i.e., not followed by storage in liquid nitrogen, markedly affected shoot tip survival. After a 30 min exposure, regrowth percentage of shoot tips decreased from 94 % (control) to 57 %, and dropped to 15 % when the treatment was prolonged up to 60 min. After a 90 min exposure, no regrowth occurred. In addition, plantlets regenerated from shoot tips which underwent 60 min or more exposure to PVS2 showed signs of malformation. Microscope observations of shoot tips treated with 0.3 or 0.4 M sucrose and 30 min PVS2 showed the presence of cells starting to plasmolyze, localized in the area surrounding the apical meristem. A limited presence of starch grains in meristem and bract cells was also noted. However, the most conspicuous consequence of prolonged PVS2 treatment was convex plasmolysis. The phenomenon was dependent on the time of PVS2 exposure. Indeed, after a 30 min treatment, plasmolysis was minimal or absent, but it increased with longer exposure to PVS2 at 4 °C.

Anatomy and osmotic potential of the Vitis rootstock shoot tips recalcitrant to cryopreservation

This study was carried out on Kober 5BB (Vitis Berlandieri × V. riparia) grape rootstock shoot tips during the preparatory steps preceding the direct immersion in liquid nitrogen, in order to overcome until now unsuccessful cryopreservation with this species. The exposure of shoot tips to 0.3-0.4 M sucrose leads to a high cell solute concentration. The treatment with plant vitrification solution (PVS2) alone, i.e., not followed by storage in liquid nitrogen, markedly affected shoot tip survival. After a 30 min exposure, regrowth percentage of shoot tips decreased from 94 % (control) to 57 %, and dropped to 15 % when the treatment was prolonged up to 60 min. After a 90 min exposure, no regrowth occurred. In addition, plantlets regenerated from shoot tips which underwent 60 min or more exposure to PVS2 showed signs of malformation. Microscope observations of shoot tips treated with 0.3 or 0.4 M sucrose and 30 min PVS2 showed the presence of cells starting to plasmolyze, localized in the area surrounding the apical meristem. A limited presence of starch grains in meristem and bract cells was also noted. However, the most conspicuous consequence of prolonged PVS2 treatment was convex plasmolysis. The phenomenon was dependent on the time of PVS2 exposure. Indeed, after a 30 min treatment, plasmolysis was minimal or absent, but it increased with longer exposure to PVS2 at 4 °C.

Cryopreservation of white mulberry (Morus alba L.) by encapsulation-dehydration and vitrification

Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration, alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations (0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification, apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration, treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel.

A Preliminary Study on Cryopreservation Protocol Applicable to all Types of Diospyros Kaki Thunb.

ABSTRACTDiospyros kaki Thunb. was divided into four types based on astringency in the fruit at harvest, presence of seed, and flesh colour: The factors that affected cryopreservation of D. kaki Thunb. were studied by modified droplet vitrification: preculture condition and PVS2 treatment time. After cryopreservation, different concentration of Pluronic F-68 in the regenerating medium was also investigated. A suitable protocol was established: shoot tips of axillaries were precultured on MS (1/2) medium with 0.5mol/L sucrose and 10g/L Pluronic F-68 for 5 days, which were loaded in loading solution for 20 min at 20°C, and incubated in PVS2 for 1.5 h at 0°C prior to direct plunge into liquid nitrogen. After rapid thawing at 40°C warm water bath and rinsed, the shoot tips were plated on the regeneration medium supplemented with 1.0mg/L TDZ, 0.6g/L PVP, 0.1% Pluronic F-68, 30g/L sucrose and 7g/L agar for one week in dark prior to exposure to normal conditions. Using this procedure in cryopreservation of D. kaki Thunb., the best regeneration rate was 80%, the average rate was 73% and the difference of four types was not significant at P=0.05.

CRYOPRESERVATION OF STRAWBERRY CV. 'SENGA SENGANA' BY DROPLET VITRIFICATION

Droplet-vitrification method was applied to shoot tips of micropropagated Fragaria ×ananassa Duch. cv. ‘Senga Sengana’. Shoot tips of 2-3 mm in length were precultured in sucrose enriched media for 24 and 48h. Subsequently, shoot tips were transferred into 6 µl droplets of PVS2 vitrification solution for 20 min and plunged afterwards into liquid nitrogen. The effect of sucrose concentration (0.1, 0.25, 0.5, 0.75, 1.0 M) used in preculture of the shoot tips regarding their regrowth, histological changes and the morpholocical stability of the regenerants in the field was analysed. The highest recovering rate (67.7%) was achieved after a preculture with 0.25 M sucrose for 24h. Recovered shoots started to multiply considerably enhancing thereby the number of plantlets ready to transfer for acclimatisation. For field evaluation 22 to 165 plants per sucrose concentration were evaluated. The number of off-types was affected by the sucrose concentration used in preculture. Light and electron microscopy revealed starch accumulation in meristematic cells after preculture in sucrose. Ultra-structure of the meristematic cells was severely affected by PVS2 and liquid nitrogen treatment.

Optimisation of Cryopreservation Technique in Mokara Golden Nugget Orchid Using PVS2 Vitrification

The study was conducted to evaluate the effects of various sucrose concentrations on Mokara orchid shoots (0.5-1.0 and 1.0-1.5 cm) after precultured at 24 and 48 h. The 1.0-1.5 cm of Mokara shoots preculture at 0.25 M sucrose for 48 h was the best condition based on TTC assay and subsequently used for the following PVS2 vitrification treatment. The selected Mokara orchid shoots were subjected to PVS2 treatment at different time of exposure (min) and temperatures (0 and 24°C). The results showed that viability of shoots reached the highest absorbance value at 10 min and 24°C. However, for the overall treatment with the results shown that 0°C temperature treatment gave the higher absorbance value which could reduce the injurious effects of PVS2. For chlorophyll determination, cryopreservation of shoots at 0°C without LN (LN-) for 5 min exposure to PVS2 recorded as highest chlorophyll content. The result also shows that total chlorophyll a for shoot in all treatment were higher than chlorophyll b.

Application of vitrification-derived cryotechniques for long-term storage of poplar and aspen (Populus spp.) germplasm

The application of three different vitrification-based freezing strategies for the cryostorage of white poplar (Populus alba L.) and hybrid aspen (P. tremula L. × P. tremuloides Michx.) have been assessed. The PVS2 vitrification protocol was successfully applied to two white poplar in vitro clones stored for more than 6 months in slow-growth conditions (4 °C, in darkness) and showing clear signs of explant etiolation and decay. After 60 min of PVS2 treatment, P. alba L. (cv. Villafranca) explants isolated from axillary buds demonstrated significantly better potential for post-freeze regrowth (64%) compared to those obtained from apical buds (17%). Similarly, a high level of survival (78%) of the frozen hybrid aspen shoot tips was recorded following the application of the same technique. Using the ‘encapsulation-vitrification’ procedure, no toxic effects of the PVS2 treatment were noticed after 120 min exposure, however none of the cryopreserved (poplar and aspen) explants survived after 3 weeks. In contrast, the ‘droplet-vitrification’ technique appeared to be very efficient in the cryopreservation of white poplar shoot tips, which increases the opportunities for wider application of this method in other woody species.;

78. Improvement of potato cryopreservation for the long-term conservation of Andean landraces at the International Potato Center (CIP)

conservation of Andean landraces at the International Potato Center (CIP). Ana Panta , Bart Panis , Cecilia Ynouye , Bram Criel , Rony Swennen , William Roca , a Laboratory of Tropical Crop Improvement, Katholic University of Leuven, 3001 Leuven, Belgium; b Genetic Resources Conservation and Characterization Division, International Potato Center, 12 Lima, Peru; c Laboratory of Proteomics, Centre de Recherche Public ’’Gabriel Lippmann’’, L-4422 Belvaux, Luxembourg The International Potato Center (CIP), Lima, Peru, conserves the world’s largest collection of potato genetic resources in vitro (4,421 cultivars belonging to 8 species). The development of new more efficient cryopreservation methods was urgently needed because the existing protocols were developed for only a very limit number of genotypes or species and were not applicable to the wide range diversity of Andean potato cultivars. Our experimental setup was based on reported relationships between cryopreservation and drought tolerance. Therefore, 4 cultivars showing different drought responses were selected for testing (Ccompis, Desiree, Pinaza and Wila Yari). A procedure originally designed for cryopreserving banana (Panis et al., 2005) was modified for potato apical shoot tips. It uses 2–3 mm shoot tips from 3 weeks old plantlets, 15–20 min exposure to a loading solution (LS), PVS2 treatment at 0–4 C, and ultra-rapid freezing in plastic straws (3 mm diameter · 2.5 cm length). After storage in liquid nitrogen, samples were thawed in MS salts enriched with 1.2 M sucrose and kept for 20 min. Shoot tips were cultivated on meristem medium (MS salts, 0.04 mg/l kinetin, 0.1 mg/l gibberelic acid) and 0.3–0.1 M sucrose concentrations, lowering the sucrose 0.1 M per day. Survival and recovery was evaluated 45 days after thawing. The efficiency of this protocol was compared with the method originally applied at CIP (Steponkus et al., 1992). For this, shoot-tips were exposed to different vitrifying solutions (Steponkus and PVS2) for 0, 10, 20, 30, 40, 50, 60, and 70 min. Exposure of 40–60 min to PVS2 showed the post-thaw highest recovery. Then, two methods to obtain ultra-rapid freezing and thawing rates were compared, i.e., loading in (1) plastic straw and (2) on aluminium foil strips. Post-thaw recovery rates were genotype dependent, varying from 47% for Desiree to 8% for Wila Yari, but independent of the freezing method. Finally, the effect of cold and sucrose pretreatments on the cryoability was evaluated. Plantlets were cultured during 3 weeks at 6 and 22 C in media containing 0.3 or 0.07 M sucrose. We observed that in the case of potato, a cold treatment increases the postthaw recovery rate for all four cultivars under investigation while a sugar treatment had no or even a negative effect. Analyses are now taking place to correlate the response towards cryopreservation with drought tolerance. Based on the results described in this report, CIP is currently applying the PVS2 droplet vitrification protocol routinely. As such, 150 accessions are now safely cryopreserved for the long-term. (Conflict of interest: None declared. Source of funding: None declared.)

The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1−1 3,6-dichloro-o-anisic acid, 0.1 mg 1−1 naphthaleneacetic acid, 2.0 mg l−1 6-benzylaminopurine, 80.0 mg 1−1 adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.

Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae

One millimeter sized shoot-tips excised from rooted in vitro plants of the genera Musa and Ensete were successfully cryopreserved with the droplet vitrification technique. We show that the loading phase can be prolonged up to 7 h and that the optimal length of PVS2 treatment is 30–50 min at 0 °C. Ultra rapid freezing and thawing rates proved to be essential for high and reproducible post-thaw regeneration rates. This results in an increase of 23–46% compared to the normal cryovial freezing protocol. When the optimal procedure was applied to 56 accessions belonging to eight different genomic groups of Musa spp. and one Ensete spp., an average of 52.9% post-thaw regeneration was obtained. These results were relatively genotype independent. Only wild diploid Musa acuminata accessions proved to be somewhat more recalcitrant towards cryopreservation though an acceptable average regeneration rate of 39% was still obtained.

CRYOPRESERVATION OF PLANT GERMPLASM

CRYOPRESERVATION OF PLANT GERMPLASM