Preculture Duration Research Articles

Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane

An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant. Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA(®) and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10min and 3min vacuum infiltered in 100 µM acetosyringone and 0.1% Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4% transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2months.

Duration of sucrose preculture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration

A simple and efficient cryopreservation protocol using encapsulation-dehydration was established for in vitro-grown shoot-tips of apple ‘Gala’ (Malus × domestica Borkh.). Shoot-tips, of 2.0 mm in length and with 5–6 leaf primordia, excised from 4-week-old shoot stock cultures, without cold-hardening, were encapsulated into beads, each being about 5 mm in diameter and containing a single shoot-tip. The beads were precultured on MS medium containing 0.5 M sucrose for 7 days. The precultured beads were dehydrated by air-drying to reduce the water content of the beads to about 22–20 % in 5–7 h, followed by a direct immersion in liquid nitrogen for 1 h. Frozen shoot-tips were re-warmed in a water bath at 38 °C for 2 min and post-cultured on a recovery medium for shoot regrowth. This protocol was successfully applied to four Malus species and one hybrid, among which M. micromalus and M. robusta are wild species native to China. The highest and lowest shoot regeneration rates were found in ‘Gala’ (75 %) and ‘Wangshanhong’ (36 %), with a mean shoot regrowth rate of 61 % attained for the seven Malus genotypes tested. Histological studies revealed that shoots could be regenerated in cryopreserved shoot-tips only when many cells in the leaf primordia and most of the cells in the apical dome survived following cryopreservation. Morphologies of the regenerated plantlets were identical to those from the in vitro stock cultures. Therefore, the encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.

Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants

An efficient transformation protocol was developed for Agrobacterium-mediated transformation of Phellodendron amurense Rupr. for using explants from mature seeds. The binary vector pCAMBIA1303, which contained hygromycin phosphotransferase (hptII) as a selectable marker gene and β-glucuronidase (GUS) as a reporter gene, was used for transformation studies. Different factors that affect survival of transformed buds, namely Agrobacterium infection method, bacterial strain, pre-culture duration, acetosyringone concentration, co-culture duration, and co-culture temperature were examined and optimized for transformation efficiency on the basis of GUS staining of hygromycin-resistant buds. Polymerase chain reaction (PCR), Southern blot and reverse transcription PCR confirmed the presence of the GUS gene. A transformation frequency of 13.1 % was achieved under optimized conditions for transformation (A. tumefaciens strain EHA105, 4 days co-cultivation at 4 °C, and infection of the pre-cultured mature-seed explants for 2 days). This is the first report of a successful genetic transformation protocol for P. amurense.

Impact of pre-culture on short- and long-term in vitro recovery of the biosynthetic potential and enzymatic and non-enzymatic antioxidant defense of Hypericum rumeliacum Boiss. after cryostorage

Due to its high hypericin and pseudohypericin in vitro biosynthetic capacity, the Balkan endemic Hypericum rumeliacum was selected as a prospective candidate for long-term preservation of valuable medicinal plant germplasm. Initial cryopreservation experiments were previously conducted based on the successful protocol established and reported for the widely studied H. perforatum. This is the first report on the impact of pre-culture duration on the short- and long-term in vitro recovery of the biosynthetic potential and antioxidant defense system of H. rumeliacum cryopreserved by vitrification. Cryopreservation did not impair the phenolics and flavonoids production of the regenerated plants. Moreover, hypericin and pseudohypericin levels even increased substantially in one of the regenerated lines, reaching yields from 0.107 and 0.752 mg g−1 DW in the control up to 0.277 and 1.112 mg g−1 DW for hypericin and pseudohypericin, respectively. However, the physical injury stress of the pre-culture treatment manipulations affected the physiological status of regenerants in a time dependent manner. Within 6 months after thawing, regenerants with the highest oxidative stress after pre-culture, were characterized with an augmentation of antioxidant metabolites such as phenolics, flavonoids, glutathione and ascorbic acid as well as increased antioxidant enzymatic activities in comparison with both the non-frozen control and the regenerants with the lowest pre-culture oxidative stress. Then, after 18 months of recovery, the same first H. rumeliacum group displayed a marked drop of enzymatic antioxidant activity as compared with the other groups of plants. Further research is needed to target oxidative stress alleviation to optimize H. rumeliacum cryopreservation protocol.

Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium-mediated transformation of tomato

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog’s (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l−1 thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l−1 in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.

Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration. Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology, particularly in organ printing. Two simple methods, hanging drop (HD) and conical tube (CT) were utilized to prepare cell aggregates. The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared. The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated. In both methods, the optimum average size of the aggregates was less than 500 microm. CT aggregates were smaller than HD aggregates. 5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface. The proliferative ability reduced when the initial cell density was increased. Comparing these methods, we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability, spreading, and proliferation. In conclusion, smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.

Long-term dynamic loading improves the mechanical properties of chondrogenic mesenchymal stem cell-laden hydrogel

Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering given their ability to undergo chondrogenesis in 3D culture systems. Mechanical forces play an important role in regulating both cartilage development and MSC chondrogenic gene expression, however, mechanical stimulation has yet to enhance the mechanical properties of engineered constructs. In this study, we applied long-term dynamic compression to MSC-seeded constructs and assessed whether varying pre-culture duration, loading regimens and inclusion of TGF-beta3 during loading would influence functional outcomes and these phenotypic transitions. Loading initiated before chondrogenesis decreased functional maturation, although chondrogenic gene expression increased. In contrast, loading initiated after chondrogenesis and matrix elaboration further improved the mechanical properties of MSC-based constructs, but only when TGF-beta3 levels were maintained and under specific loading parameters. Although matrix quantity was not affected by dynamic compression, matrix distribution, assessed histologically and by FT-IRIS analysis, was significantly improved on the micro- (pericellular) and macro- (construct expanse) scales. Further, whole genome expression profiling revealed marked shifts in the molecular topography with dynamic loading. These results demonstrate, for the first time, that dynamic compressive loading initiated after a sufficient period of chondro-induction and with sustained TGF-beta exposure enhances matrix distribution and the mechanical properties of MSC-seeded constructs.

Optimization and Transformation of Garden Balsam, Impatiens balsamina, Mediated by Microprojectile Bombardment

In this study, a transformation system was developed by initially optimizing the microprojectile bombardment parameters for cotyledonous explants. The parameters optimized were combination of distance from stopping screen to target tissue and helium pressure, number of bombardment, preculture duration prior to bombardment, DNA amount, osmoticum treatment and post-bombardment incubation time. Determination of minimal inhibitory concentration for efficient selection of transformants was also carried out for hygromycin. Different concentrations of hygromycin (25. 50, 75 and 100 mg L-1) were tested against different ages of explants (0, 1, 3 and 5 weeks) for efficient selection of transformants. Using the optimized parameters, transformation of cotyledons was carried out followed by selection on 75 mg L-1 hygromycin at different weeks of post-bombardment. Transgenic plants were successfully regenerated from bombarded explants and confirmed via histochemical GUS assay and PCR analysis.

Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat

Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase II, (npt II) and beta-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23-25 degrees C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23-24 degrees C for 2-3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.

CRYOPRESERVATION OF EMBRYOGENIC SUSPENSION CULTURES OF CYCLAMEN PERSICUM MILL.

We have developed a protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum, which does not involve the need of sophisticated equipment. With the aim of achieving this simple protocol for cryopreservation, different cryoprotectants in the pre-culture and pre-treatment periods on cell growth and re-growth after cryopreservation were tested. For cryopreservation experiments embryogenic suspension cultures in the linear growth phase 7-10 days after subculture were used. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rate of 75% based on the number of Petri dishes plated after thawing. Additionally, a pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h immediately prior to freezing, also positively affected re-growth. Experiments in which the optimal pre-culture duration was varied showed that 2-4 days was the best exposure time to 0.6 M sucrose. Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen control callus. Microscopic studies on viability using FDA (fluorescein diacetate) staining revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died.

Efficient shoot regeneration andAgrobacterium-mediated transformation ofBrassica juncea

Procedures for callus induction, plantlet regeneration, andAgrobacterium-mediated transformation ofBrassica juncea were optimized by studying several factors, including explant types, and various plant growth regulators and adjuvants, such as silver nitrate, sucrose and agar. The highest shoot regeneration frequency was obtained from hypocotyl and cotyledonary petiole explants on MS medium containing 3 mg/L benzylaminopurine (BA) and 2 mg/L α-naphthaleneacetic acid (NAA). Transformation was affected by a number of factors, including explant type, selection agents, preculture duration, pre-selection conditions, and coculture temperature. Transformation efficiencies for hypocotyl and cotyledonary petiole explants were at 65% and 69%, respectively.

Optimization of Dynamic Culture Conditions: Effects on Biosynthetic Activities of Chondrocytes Grown in Collagen Sponges

Application of mechanical stimulation, using dynamic bioreactors, is considered an effective strategy to enhance cellular behavior in load-bearing tissues. In this study, two types of perfusion mode (direct and free flow) are investigated in terms of the biosynthetic activities of chondrocytes grown in collagen sponges by assessment of cell proliferation rate, matrix production, and tissue morphology. Effects of the duration of preculture and dynamic conditioning are further determined. Our results have demonstrated that both bovine and human-derived chondrocytes demonstrate a dose-dependent response to flow rate (0-1 mL/min) in terms of cell number and glycosaminoglycan (GAG) content. This may reflect the weak adhesion of cells to the sponge scaffolds and the immature state of the constructs even after 3 weeks of proliferative culture. Our studies define an optimal flow rate between 0.1 and 0.3 mL/min for direct perfusion and free flow bioreactors. Using fresh bovine chondrocytes and a lower flow rate of 0.1 mL/min, a comparison was made between free flow system and direct perfusion system. In the free flow bioreactor, no cell loss was observed and higher GAG production was measured compared with static cultured controls. However, as with direct perfusion, the enhancement effect of free flow perfusion was strongly dependent on the maturation and organization of the constructs before the stimulation. To address the maturation of the matrix, preculture periods were varied before mechanical conditioning. An increase in culture duration of 18 days before mechanical conditioning resulted in enhanced GAG production compared with controls. Interestingly, additional enhancement was found in specimens that were further subjected to a prolonged duration of perfusion (63% increase after an additional 4 days of perfusion) after prematuration. The free flow system has an advantage over the direct perfusion system, especially when using sponge scaffolds, which have lower mechanical properties; however, mass transfer of nutrients is still more optimal throughout the scaffolds in a direct perfusion system as demonstrated by histological analysis.

Calluses initiated from thin mature embryo fragments are suitable targets for wheat transformation as assessed by long-term GUS expression studies

Microprojectile- or Agrobacterium-mediated DNA delivery into calluses initiated from immature embryos has proven to be effective in transforming wheat. Yet, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process. To circumvent these limitations, we propose an alternative technique applying the particle bombardment technology to calluses derived from fragmented mature embryos rather than immature tissues. The phosphinothricin acetyl transferase (bar) and β-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimise the performance of the proposed technique. Primary requirement for genetic transformation method development, the regeneration capacity of bombarded calluses was established. A preculture duration of 6 days was identified as optimal for DNA uptake and β-glucuronidase (GUS) expression. The highest activity was recorded when calluses were selected. Long-term GUS expression studies (1–7 weeks subsequent to bombardment), showed differentiated behaviours for tissues obtained from mature versus immature embryos. Notably, mature embryos exhibited the greatest number of cells stably expressing the reporter gene, thus providing an excellent source material for developing a stable transformation procedure.

A simple and efficient cryopreservation of in vitro -grown shoot tips of `Troyer' citrange [ Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-vitrification

A simple and efficient method was developed for cryopreservation ofin vitro-grown shoot tips of `Troyer' citrange byencapsulation-vitrification. Excised shoot tips were precultured withincreasing sucrose concentrations of 0.3, 0.5, 0.75 and 1 M for 4 days.Precultured shoot tips were encapsulated and simultaneously osmoprotectedwith a loading solution of 2 M glycerol and 1 M sucrose.Osmoprotected and encapsulated shoot tips were dehydrated with a highlyconcentrated vitrification solution prior to direct immersion in LN for 1 h.Optimal survival of cryopreserved shoot tips was obtained when preculturedshoot tips were osmoprotected for 60 min during encapsulation.Dehydration by exposure to modified PVS2 solution for 90 min at24 °C and for 180 to 210 min at 0 °C was found optimalfor survival of cryopreserved shoot tips. Preculture duration largelyinfluenced survival of cryopreserved shoot tips, with the best result obtainedafter 2 to 5 days of preculture with stable 1 M sucrose. With theoptimized parameters, 100% survival of cryopreserved shoot tips wasachieved. Morphologies of plants regenerated from cryopreserved shoottips were similar to those of the seedlings.

Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue.

A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated. The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here. The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density. Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated. Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression. The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.

Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol

An efficient vitrification procedure was developed and successfully applied to cryopreserve six endangered West Australian species (family Haemodoraceae: Anigozanthos humilis ssp. chrysanthus Hopper;A. kalbarriensis Hopper;A. viridis ssp. terraspectans Hopper;Conostylis dielsia ssp.teres Hopper;C. micrantha Hopper and C. wonganensis Hopper). Species were initially evaluated for cryostorage using a basic vitrification protocol involving: culturing plantlets in vitro for 21d; excision of shoot apices; preculture of apical tips on 0.4M sorbitol for 2d, followed by incubation in PVS2 (plant vitrification solution 2) for 25min at 0°C, then direct immersion in liquid nitrogen (LN). Warming of retrieved material was for 1min in a 40°C water bath. Using this protocol five of the six species exhibited low post-storage survival, while the sixth species, A. viridis ssp. terraspectans posted higher survival (61.1%). Using A. viridis ssp. terraspectans as an indicator species, the initial protocol was modified to include: 3d preculture on 0.80M glycerol, loading treatment with 2.0M glycerol plus 0.4M sucrose solution for 20min, followed by 25min exposure to a modified PVS2. Survival was significantly improved in the test species, and in further experiments three other species also showed significant improvements with the new protocol. Key findings include: effectiveness of glycerol in the preculture medium; the effect of preculture duration; the importance of a loading stage for these species; and the successful use of modified PVS2 solutions with reduced or zero dimethyl sulfoxide (DMSO).

Role of intracellular water retention strength in freezing tolerance of Chrysanthemum cinerariaefolium Vis. cell cultures

Summary An efficient procedure using sucrose as cryoprotectant to confer freezing tolerance on high-pyrethrinproducing cell lines of Chrysanthemum cinerariaefolium was developed. The efficiency of sucrose depended on both its concentration in the pregrowth medium and on preculture duration. A low sucrose concentration (0.25 mol/L) induced insufficient dehydration of cells, and so failed to prevent damage during freezing and prevented regrowth. The high cell dehydration obtained by incubating cells for 10 days in a medium supplemented with 0.5 mol/L sucrose was essential for freezing tolerance, but was insufficient to induce cell cryoprotection on its own. At this concentration, the post-thaw viability race increased with culture duration. Twenty days of pretreatment afforded a 57% viability rate, which was correlated with a different partitioning of water into unfrozen and frozen fractions, without modifications in cell dehydration. To study the water sorption strengths in pretreated cells, sorption isotherms were constructed. The effects of sucrose concentration and preculture duration during pretreatments were investigated. Sorption isotherms were analysed according to the D'Arcy and Watt model. Sucrose acted by trapping intracellular water through modification of hydrogen and multimolecular linkages, and thus increased the unfrozen water fraction in cells.

Genetic transformation of Linum by particle bombardment

Linseed flax (Linum usitatissimum L.) was transformed by bombarding hypocotyl tissues with gold particles coated with plasmid DNA carrying the β-glucuronidase (GUS) (uid-A) and neomycin phosphotransferase II (npt-II) genes. Transient expression of the introduced β-glucuronidase gene was used to study factors influencing the DNA delivery, while progeny analyses confirmed stable transformation. The efficiency of DNA delivery, uptake and expression was significantly affected by the duration of hypocotyl preculture, bombardment distances, the level of chamber vacuum, the quantity of DNA, and the size of particles. Nineteen independent GUS-positive shoots were recovered and regenerated into whole plants, from which 10 plants successfully produced viable seeds. Analysis of T1 and T2 self pollinated progeny for histochemical and fluorometric GUS assays and polymerase chain reaction (PCR) analyses for uid-A, plus npt-II PCR and germination assays in progeny plants demonstrated that the transgenes were expressed in selected plants and transmitted to progeny, usually via a single Mendelian locus. The results show that particle bombardment can be used to produce transgenic Linum plants. The system is rapid, simple and offers an alternative to Agrobacterium methods.

Cryopreservation of potato shoot tips by encapsulationdehydration

Potato shoot tips excised from 2-week-old in vitro nodal cuttings were cryopreserved after encapsulation in alginate beads. Encapsulated shoot tips were first precultured in sucroseenriched media, dried over silica gel, and rapidly cooled in liquid nitrogen. After slow rewarming in air at room temperature, alginate beads were transferred to solid culture medium for shoot tip recovery. After cooling in liquid nitrogen, shoot yield depended on preculture duration, sucrose concentration and water content of beads. Survival rates above 60% were obtained for each cultivar tested.