Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Cryopreservation of in vitro-grown apical meristems of Lilium by droplet-vitrification

In this study, apical meristems from adventitious buds of three lily (Lilium L.) cultivars were successfully cryopreserved by droplet-vitrification. The most effective techniques were as follows. Excised meristems from in vitro plantlets which had been sub-cultured for about 2 months were cold-hardened at 4 °C for 1 week, precultured on MS medium supplemented with 0.3 mol L⁻¹ sucrose for 2 days, osmoprotected in loading solution for 20-40 min at room temperature and then soaked in PVS2 solution for 90-120 min at 0 °C, frozen in microdroplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. The meristems were then rapidly rewarmed by dilution solution, transferred to regeneration medium and stored in the dark for two weeks at 20 °C, and then cultured under white fluorescent light at an intensity of 2000 lux, with a 16 h photoperiod at 20 °C. The highest post-thaw survival percentages of three cultivars ‘Siberia’ (Lilium × siberia), Lilium lancifolium Thunb. and ‘Snow Queen’ Lilium × longiflorum were 65.0%, 83.8% and 43.3%, and regeneration percentages were 62.0%, 67.6% and 35.0%, respectively. The study demonstrated that cryopreservation by droplet-vitrification increased survival and regeneration percentages of certain lily cultivars compared with vitrification. Thus to cryopreserve lily meristems, droplet-vitrification method is preferable to the vitrification method.     

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

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 in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification

 Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm. Received: 24 November 1998 / Revision received: 8 February 1999 / Accepted: 26 February 1999     

The effect of cadmium on shoot apical meristems of barley

We studied the effects of cadmium acetate at various concentrations (200 to 800 mg/kg substrate) on growth and development of shoot apical meristem of the barley plants (Hordeum vulgare L.) under the conditions of vegetative experiment. It was shown that in the presence of increasing cadmium concentrations in the soil substrate, the apex length and number of inflorescence elements decreased and the rate of organogenesis slowed down, thus affecting the spike potential productivity and morphological parameters of barley plants at the flowering stage. It is possible that the negative effect of cadmium on the shoot apical meristem is associated with its influence on division of the apex cells.     

Cryopreservation of immature seeds of Bletilla striata by vitrification

An efficient protocol was established for the cryopreservation of immature seeds of a terrestrial orchid, Bletilla striata. Immature seeds collected 2–4 months after pollination (MAP) were treated using three different cryogenic procedures: (1) direct plunging into liquid nitrogen, (2) vitrification, and (3) vitrification with preculture. When immature seeds collected 3 MAP and 4 MAP were precultured for 3 days on New Dogashima medium supplemented with 0.3 M sucrose and cryopreserved by vitrification, the survival rate after preservation, as assessed by staining with 2,3,5-triphenyltetrazolium chloride, was 92% and 81%, respectively. Immature seeds thus treated showed no decrease in germination rate relative to untreated immature seeds, and they developed into normal plantlets in vitro.     

Cryopreservation of Hyoscyamus niger adventitious roots by vitrification

An auxin-independent adventitious root culture of Hyoscyamus niger was established, and the roots were successfully cryopreserved with a high regeneration rate of 93.3 percnt; by vitrification method. The root tips were cultured for 12 to 14 days in phytohormone-free Murashige and Skoog (MS) liquid medium, and were excised and precultured on Woody Plant (WP) solid medium supplemented with 0.3 mol/L sucrose at 25 °C in the dark. After 1 day, they were treated with MS-based loading solution for 10 min, followed by soaking in MS-based PVS2 for 10 min at 0 °C. The treated root tips were immersed in liquid nitrogen (-196 °C). For recovery, the root tips were thawed rapidly at 40 °C and washed with MS medium containing 1 mol/L sucrose prior to plating onto WP solid medium. The regenerated roots were evaluated by their growth and tropane alkaloid production. The growth and alkaloid content of regenerated roots analyzed using HPLC were found to be almost the same as those of non-treated roots.     

Invasion of American native lily populations by an alien beetle

The lily leaf beetle (Lilioceris lilii), indigenous to Eurasia, was first detected in North America on the island of Montréal, Québec, Canada in 1943. Populations remained limited to the original site of introduction but since 1978 the distribution of this species has expanded in all directions. To date L. lilii has only been recorded feeding on cultivated lilies in urban and rural areas. But as the beetle spreads its geographic range it could encounter native lily species. The objectives of this study were to determine the suitability of two indigenous North American lily species (Lilium canadense and L. philadelphicum) as host for L. lilii, and to assess the degree of infestation of wild lily populations. Measurement of fitness parameters under laboratory conditions (survival, development time, size) indicated that L. lilii has the capacity to develop on both species of native lilies. In the provinces of Québec and Ontario eight of the 20 wild populations of L. canadense sampled were infested. In contrast, L. philadelphicum was not attacked by the alien beetle. A number of native lilies are already endangered in North America, so there is an urgent necessity to address the ecological impact of L. lilii in natural ecosystems.     

Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development of a microtargeting device for particle bombardment of plant meristems

A gene transfer system for meristem cells was developed on the basis of a ballistic approach. In order to meet some important prerequisites for an efficient transfer system, such as for example aiming at small tissues and control of penetration of the microprojectiles, we developed an acceleration system fundamentally different from the usual macroprojectile driven approach. Instead of a macroprojectile, microtargeting uses the law of Bernoulli for accleration of highly uniform-sized gold particles. The system is able to deliver 80% of the particles to an area as small as 150 micron in diameter, which corresponds to the size of a meristem. Microtargeting yields gene delivery (measured as number of transiently GUS expressing cells to up to 3% of the cells exposed in the target area or up to 35 × 10³ cells per cm². Stable transformation of tobacco microcolonies with the microtargeting device was shown to have an efficiency up to one stable transformant per 1000 cells exposed to the shot, or up to one transformant per shot. We perform 4 or 5 shots per min. After 30 to 40 shots, reloading can take up to 2 min. Microtargeting is very flexible and allows for the adjustment of the important parameters to fit the requirements of the respective tissue.     

Graft and dodder transmission of phytoplasma affecting lily to experimental hosts

Experimental infection of Alstroemeria seedlings with naturally infected lily ‘Casablanca’ with stunting and flower bud deficiency phytoplasma resulted 3–4 weeks after top grafting in chlorotic and/or necrotic stripes, whitening of the leaves, shoot necrosis and die back. Flower discoloration or malformation were not observed. Attempts to transmit phytoplasma from naturally infected lily and experimentally infected Alstroemeria to Catharanthus roseus by top grafting resulted in stunted growth, dull yellowing and malformation of the leaves in 4–6 weeks. Some plants were temporary entirely vegetative and did not produce flowers. The periwinkle plants that were bridged by Cuscuta odorata from the diseased lilies and Alstroemerias showed similar symptoms as top-grafted ones. With the universal primer pairs rU3/fU5 specific PCR product with expected length ∼900 was amplified from samples collected from lilies with severe symptoms and top grafted test plants. All PCR products used for RFLP analysis after digestion with Alu I showed the same restriction profiles. Position of three obtained bands corresponded to the lengths of the DNA fragments of American aster yellows (AAY) phytoplasma group.     

卵叶泥炭藓茎尖包埋玻璃化法超低温保存研究

该研究通过对脱水时间和化冻温度的探索,检验了包埋玻璃化法在超低温保存湿润生境中苔藓的可能性。结果表明:卵叶泥炭藓无菌苗在4℃条件下预培养3d后,在0℃用60% PVS_2装载30min,PVS_2脱水60min后迅速投入液氮保存,24h后用40℃水浴快速化冻2min再培养,成活率可达42.41%,且再生植株与常温状态下的植株形态指标没有显著性差异。研究认为,包埋玻璃化法超低温保存湿润环境中生长的苔藓植物是可行的。     

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     

Development of dormancy in different lily genotypes regenerated in vitro

Dormancy development in four Lilium genotypes,L. speciosum, Star Gazer, C. King and Snow Queenregenerated in vitro was compared. Major factorsinfluencing dormancy development were the same for different genotypes andespecially L. speciosum and Star Gazer, that are closelyrelated, reacted similarly. Temperature was the main factor in dormancyinduction and breaking. The range of temperatures that induced dormancy and thelevel of dormancy that developed differed per genotype. In Star Gazer, dormancydeveloped gradually but in Snow Queen, dormancy developed very fast. Thereactions to temperature, reflected the climate in the area of origin. Abscisicacid deepened the level of dormancy induced by temperature but had no effectunder non-inductive temperature conditions. When abscisic acid synthesis wasblocked, no dormancy developed. Dormancy in all genotypes was broken by coldincubation for severalweeks. The cold requirement of the genotypes differed in line with the naturalwinter conditions in their habitat. The effect of hormones on dormancy breakingwas also investigated. A gibberellin treatment of 24 h brokedormancy in L. speciosum, Star Gazer and Snow Queen.     

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     

Plantlets from encapsulated meristems of Gentiana pneumonanthe L.

The preparation of synthetic seeds of marsh gentian from apical and axillary meristems, using 0.1 M CaCl₂ and 3 % solutions of sodium alginate (aquatic solution, aquatic with IAA, and with MS medium) and the results of regrowth of these seeds are discussed. The best regeneration occurred in the case of apical meristems encapsulated in 3 % sodium alginate prepared on the basis of MS medium.     

Root apical meristems ofAllium porrum L. as affected by arbuscular mycorrhizae and phosphorus

Summary Arbuscular mycorrhizal (AM) fungi significantly improve plant growth in soils with low phosphorus availability and cause many changes in root morphology, similar to those produced by increased P nutrition, mainly depending on root apex size and activity. The aim of this work was to discriminate between the morphogenetic role of AM fungi and P in leek (Allium porrum L.) by feeding mycorrhizal and nonmycorrhizal plants with two nutrient solutions containing 3.2 or 96 M P and examining specific parameters related to adventitious root apices (apex size, mitotic cycle, and RNA synthesis). The results showed that AM fungi blocked meristem activity as indicated by the higher percentages of inactive apices and metaphases in the apical meristem of mycorrhizal plants, whereas the high P supply lengthened the mitotic cycle without blocking the apices, resulting in steady, slow root growth. The possible involvement of abscisic acid in the regulation of root apex activity is discussed.Abbreviations ABA abscisic acid - AM arbuscular mycorrhizae - CI and CII nonmycorrhizal control plants grown with low or high phosphorus concentration - MI and MII mycorrhizal plants grown with low or high phosphorus concentration - PGR plant growth regulator