High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

An efficient,widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg/l α-naphthaleneacetic acid and 0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length) including 2–3 leaf primordia were precultured on Murashige and Skoog (MS; 1962) medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µl PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for shoot regrowth. Shoot regrowth levels ranged from 42.5 % for L. longiflorum × Oriental ‘Triumphator’ to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1 % across the six diverse Lilium genotypes tested. Histological observations found that the survival patterns were similar in cryopreserved shoot tips of ‘Triumphator’ and ‘Siberia’. Assessments using inter-simple sequence repeat markers found no differences in regenerants recovered from the control stock cultures and from cryopreserved shoot tips in ‘Triumphator’ and ‘Siberia’. This Lilium droplet-vitrification cryopreservation method is efficient, simple and widely applicable for the long-term conservation of lily genetic resources.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Cryopreservation of isolated mint shoot tips by vitrification

Shoot tips isolated from a mint clone, Mentha aquatica x M. spicata, were gradually exposed to a mixture containing 35% ethylene glycol, 1 M dimethylsulfoxide and 10% polyethylene glycol-8000 and then immersed into liquid nitrogen. Cooling and warming rates were approximately 4800°C/min and 9000°C/min respectively. Survival after liquid nitrogen treatment ranged from 31% to 75% among experiments. There was no obvious reason for this variation. In many cases the treated shoot tip directly developed into a shoot without any or with only slight callus formation.Abbreviations DSC differential scanning calorimetry - DMSO dimethylsulfoxide - EG ethylene glycol - PEG-8000 polyethylene glycol - MW avg. 8000 - LN liquid nitrogen - IBA indolebutyric acid - BA benzyladenine     

The cryopreservation of shoot tips of Rosa multiflora

Shoot tips of in vitro grown plantlets of Rosa multiflora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.Abbreviations RSC1 modified Murashige and Skoog medium for Rosa multiflora shoot culture     

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.     

Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips

 Shoot tips from in vitro-grown, cold-hardened stock plants of white poplar (Populus alba L.) were successfully cryopreserved at –196  °C by one-step vitrification. After preculturing at 5  °C for 2 days on hormone-free MS medium containing different sucrose concentrations, and loading for 20 min with 2 m glycerol and 0.4 m sucrose, shoot tips were treated with the PVS2 vitrification solution and plunged directly into liquid nitrogen. Best survival rate (90%) was obtained when shoot tips were precultured on 0.09 m sucrose, hormone-free MS medium, vitrified by exposure to PVS2 solution for 60 min at 0  °C and, following cryopreservation, rewarmed at 40  °C and washed in 1.2 m sucrose solution for 20 min. Regrowth was improved by plating shoot tips on a gelled MS medium containing 1.5 μm N⁶-benzyladenine plus 0.5 μm gibberellic acid, while shoot rooting was achieved on MS medium containing 3 μm indole-3-butyric acid. Following this procedure, almost 60% rooted shoots were obtained from cryopreserved shoot tips. Received: 1 February 1999 / Revision received: 3 May 1999 · Accepted: 21 May 1999     

Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification

 Routine cryopreservation of shoot tips from sweet potato [Ipomoea batatas (L.) Lam] has been hampered by their survival variability after cryogenic exposure. We examined the effects of light conditions on stock plants, sucrose preculture and cryoprotectant loading on survival after vitrification using PVS2 solution. The survival of vitrified sweet potato shoot tips cooled to approximately –208  °C was increased by preculturing with 0.3 M sucrose for 24 h at 22  °C. Survival was also enhanced by excising shoot tips immediately after the 8-h dark photoperiod. The best survival after cryogenic exposure was obtained using 2 M glycerol +0.4 M sucrose for 1 h at 22  °C followed by dehydration with PVS2 for 16 min at 22  °C. Rapid cooling was used and achieved by the immersion of foil strips into partially solidified nitrogen. Successfully vitrified and warmed shoot tips directly developed shoots on a medium containing 1 μM NAA, 0.5 μM BA and 0.1 μM kinetin with only minimum callus formation. Shoot formation occurred in all surviving shoot tips. This procedure shows promise for cryopreserving sweet potato shoot tips. Received: 2 March 1999 / Revision received: 21 September 1999 / Accepted: 29 September 1999     

Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tips by vitrification

Summary Vitrification is a technically simple method for cryopreserving plant germplasm, requiring only the application of suitable cryoprotectants and rapid cooling rates. Sweetpotato (Ipomoea batatas [L.] Lam.) shoot tips obtained from in vitro plants survived liquid nitrogen (–196°C) exposure following a vitrification-inducing pretreatment. Shoot tips were treated in a stepwise manner with a vitrification solution containing 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in growth medium. Incubation of shoot tips for 1 to 2 h in low concentrations of the vitrification solution enhanced survival. Most surviving shoot tips developed callus, and a variable percentage subsequently formed shoots. Survival was not achieved using two-step cooling procedures. The percentage of shoot tips surviving vitrification and those subsequently forming a shoot varied widely among replications.Abbreviations BA N⁶-benzyladenine - IBA indole-3-butyric acid - EG ethylene glycol - DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) minerals and vitamins - LN liquid nitrogen - PI plant introduction     

Improved survival after cryogenic exposure of shoot tips derived from in vitro plantlet cultures of potato

In vitro plantlets were used for axillary shoot tip isolation to avoid microbial contamination which often occurred from use of greenhouse-grown plants. Periodic subculturing gave a supply of uniform plantlets necessary for obtaining shoot tips for cryogenic experiments. Previous results have shown that all cells within a shoot tip did not survive cryogenic exposure and the regrowth percentage depended upon the composition of the culture medium. A medium containing 0.5 mg/liter zeatin, 0.2 mg/liter gibberellic acid, and 0.5 mg/liter indoleacetic acid gave regeneration of a multiple-shoot mass from control and some frozen shoot tips. The two-step cooling procedure (0.2-03 degrees C/min to -35 degrees C followed by immersion in liquid nitrogen) gave high percentages of regrowth in the cultivars Norland and Red Pontiac. Shoots were obtained from treated materials in both cultivars.     

Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration

The aim of this study was to compare the genetic stability of chrysanthemum (cv. Pasodoble) apices cryopreserved using two different methods: encapsulation-dehydration and vitrification. The assessment of the genetic stability was developed using RAPDs markers. Assessment of stability was evaluated in pot-cultivated mother plants (from which buds were excised for micropropagation), in shoots (leave tissue) from which apices were extracted for cryopreservation, and in shoots regenerated from cryopreserved apices 30 days after recovery and after further 3 months in culture. Throughout the process the origin of the apices (in vitro-shoot from which they were excised) was recorded. Twenty one regenerants cryopreserved by vitrification and 25 by encapsulation-dehydration were assessed. Only one cryopreserved regenerant from the encapsulation-dehydration method showed a different band pattern. These results support the necessity of monitoring the genetic stability of the regenerants obtained after cryopreservation, as this is a very useful technique for the conservation of plant genetic resources.     

In Vitro Plantlet Formation in Mangosteen (Garcinia mangostana L.)

Optimum conditions were determined for in vivo growth and multiplicationof Garcinia mangostana L. using explants from aseptically germinatedseedlings and field-grown plants. Proliferating shoots wereobtained from cotyledon segments cultured on modified Murashigeand Skoog's (1962) medium with 6-benzylaminopurine. Juvenileleaf segments produced adventitious buds on Woody Plant Medium(Lloyd and McCown, 1981). Root segments gave few buds. Shoottip, nodal, and internodal explants gave multiple axillary andadventitious buds. Shoots were multiplied by enhanced axillaryand adventitious bud formation. The shoots were rooted withindolebutyric acid treatment. Rooted shoots were readily establishedin vermiculite: sand (1:1) mixture. Garcinia mangostana L., Mangosteen, tissue culture, shoot regeneration, bud development     

Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure

Invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott.) were successfully cryopreserved by vitrification. Excised shoot tips precultured on solidified MS supplemented with 0.3M sucrose and maintained under a 16 h phtoperiod at 25°C for 16 h were loaded with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. The shoot tips were then sufficiently dehydrated with a highly concentrated vitrification solution (PVS2) for 20 min at 25°C prior to immersion into liquid nitrogen. Successfully vitrified and warmed shoot tips resumed growth within 7 days and developed shoots directly without intermediate callus formation. The average rate of shoot recovery amounted to around 80%, and the vitrification protocol appeared to be very promising for the cryopreservation of taro germplasm.Abbreviations DMSO Dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS Murashige & Skoog medium (1962) - TDZ thidiazuron     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20 min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2 M sucrose solution for 20 min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10 days of plating and developed shoots within 3 weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.     

In vitro polyploidization from shoot tips of Jatropha curcas L.: a biodiesel plant

Jatropha curcas L. has been considered one of the most promising alternatives for biofuel production and, thus, a relevant economic crop. In this context, in vitro tissue culture techniques such as organogenesis and embryogenesis have been conducted for mass clonal propagation of elite J. curcas lines. However, despite advancements, in vitro induction of polyploids has not yet been related for this crop. In this sense, the present study attempted to induce polyploidy in plantlets generated from shoot tips of J. curcas ??Gon?alo?? (2n?=?2×?=?22 chromosomes, 2C?=?0.85?pg). For this purpose, some criteria were adopted for selection of the most adequate colchicine treatment: (a) survival rate of the explants, and (b) number of tetraploid and (c) mixoploid plantlets. Tetraploid and mixoploid plantlets were obtained from different treatments, with 0.5?mM colchicine for 96?h being the most efficient. The plantlets were recovered and clonally propagated in tissue culture medium supplemented with indole-3-acetic acid and 6-benzylaminopurine. These results show that the tissue culture procedures were adequate for induction, propagation and recovery of tetraploid and mixoploid plantlets. Moreover, DNA ploidy level screening by flow cytometry was a practical and rapid strategy for selection of diploid, mixoploid and tetraploid plantlets. The tissue culture system presented here represents a reliable methodology for in vitro polyploid induction of this and other elite lines of J. curcas.     

Development of infantile rat ovaries autotransplanted after cryopreservation by vitrification

We cryopreserved infantile rat ovaries by vitrification and assessed their viability by autotransplantation. Hemilateral ovarian transplantation was performed on rats on postnatal Days 10 to 12. The left ovary of each rat was dissected out, cryopreserved by vitrification using a modified vitrification solution (VS1), and then autotransplanted under the capsule of the right kidney. The right ovary of each rat was removed. For the control, the left ovary was dissected out from each rat and was immediately transplanted by the same procedure, without cryopreservation. Rats were nursed until weaning, and then the day of vaginal opening, estrous cyclicity from the day of vaginal opening until postnatal Day 84, and histology of ovarian grafts at postnatal Day 84 were examined. The time course of development of endocrine function of cryopreserved grafts was similar to that of fresh grafts. In ovarian transplants recovered on postnatal Day 84, antral follicles and corpora lutea (CL) were observed in addition to small follicles, although the number of antral follicles in cryopreserved grafts was smaller than in the fresh grafts. These results indicate that cryopreservation of ovarian tissue by vitrification can be used for the preservation of fertility and endocrine function of ovaries.     

Pollen development in male sterile mangosteen (Garcinia mangostana L.) and male fertile seashore mangosteen (Garcinia celebica L.)

Protoplasma - Mangosteen (Garcinia mangostana L.) is an economically important tropical fruit, yet the reproductive biology of this dioecious plant is complex. Male trees are not known, and female...