Traits of Panax ginseng hairy roots after cold storage and cryopreservation

Summary Panax ginseng hairy root cultures were established by infecting petiole segments with Agrobacterium rhizogenes strain 15834. Hairy root segments including root tips placed onto phytohormone-free 1/2 Murashige and Skoog solid medium and stored at 4 °C in the dark for 4 months, resumed elongation when the temperature was raised to 25 °C in the dark. For cryopreservation, a vitrification method was applied. Root tips precultured with 0.1 mg/l 2,4-D for 3 days and dehydrated with PVS2 solution for 8 minutes prior to immersion into liquid nitrogen had a survival rate of 60 % and could regenerate. The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside productivity and patterns as those of the control hairy roots cultured continuously at 25 °C. The conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.Abbreviations 1/2 MS a half strength Murashige and Skoog (1962) - B5 Gamborg B5 (Gamborg et al. 1968) - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - RCI root culture medium containing 100 mg/l myoinositol - HF phytohormone-free - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - PCR polymerase chain reaction - PVS2 plant vitrification solution 2 (Sakai et al., 1990) - FDA fluorecein diacetate     

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     

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     

蒲氏钩蝠蛾幼虫血淋巴物质与其耐寒性

作为冬虫夏草寄主,蒲氏钩蝠蛾具重要的经济研究价值,主要分布于青藏高原色季拉山,具有较强的耐寒性,其耐寒性机理尚不明了。通过调查蒲氏钩蝠蛾生境土壤温度与血淋巴物质组成周年变化,以期探讨其耐寒性机理。实验结果表明:土壤温度呈规律性升降趋势,1、2月最低,8月逐步上升至最高,而后逐渐下降;血淋巴中主要物质按含量从高到低排列依次为:蛋白质(8.37—74.2 mg/m L)、总糖(12.65—36.12 mg/m L)、脂肪(7.7—12.32 mg/m L)以及甘油(1.66—3.91 mg/m L);在温度较高的夏季,蛋白质、总糖以及脂肪等物质含量处于较低水平,相反,在寒冷的冬季,此三者的含量均居较高水平;此3类能源物质含量与野外土壤温度均呈极显著的负相关关系,因而血淋巴总热量值在夏季较低而冬季较高,与野外土壤温度亦呈极显著负相关关系;与之相反的是,血淋巴中甘油含量的周年变化规律与野外土壤温度无明显的相关关系。因此,血淋巴中蛋白质、总糖以及脂肪等物质对于蒲氏钩蝠蛾耐寒性有着非常重要的作用,幼虫通过积累3类物质,增加血淋巴能量值,抵御青藏高原的高寒气候;可能由于生境的不同,甘油在蒲氏钩蝠蛾中的作用与其它昆虫有差异,在耐寒性提高中的功能尚需进一步研究。实验结果为了解蒲氏钩蝠蛾的强耐寒性以及适应高寒环境的生理生化机制提供理论依据。     

Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communis L.)

An efficient adventitious shoot regeneration system was developed for pear (Pyrus communis L.), using leaves from in vitro proliferating shoots. Under optimal conditions, bud regeneration frequencies of Comice, Passe-Crassane, Williams and Conference ranged from 60% to 97%, with the mean number of shoots per regenerating leaf ranging from 3.2 to 6.6. Despite the great variability in responses of the different cultivars, in general an initial dark exposure of at least 20 days was required. Ammonium and total nitrogen proved to play an essential role: intermediate NH₄ ⁺ concentrations were suitable for regeneration. The balance between NH₄ ⁺ and NO₃ ^- also influenced regeneration; optimal regeneration occured on media with a 1:3 NH₄ ⁺/NO₃ ^- ratio. TDZ at 1 M was less efficient than higher concentrations, whatever the NAA level. Finally, length and growth regulator composition of the two phases (induction and expression) influenced the regeneration rate of Conference.Abbreviations BA 6-benzyladenine - EDFS ethylenediamine-tetraacetic acid ferric-sodium salt - IBA 4-indole-3yl-butyric acid - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea)     

Adventitious shoot regeneration from vegetative shoot apices in pear and putative role of cytokinin accumulation in the morphogenetic process

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 M 6-benzyladenine, 1.0 M -naphthaleneacetic acid and 250 mg l^–1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.     

Inheritance of the photoperiodically induced cold acclimation response in Cornus sericea L., red-osier dogwood

Summary Cold acclimation responses of latitudinal ecotypes of Cornus sericea L. (C. stolonifera Michx.) and F₁, F₂ and BC₁ hybrid progenies were measured under natural photoperiod conditions in St. Paul, MN and artificially shortened photoperiods in the glass-house. The 65 °N and 62 °N ecotypes (Alaska and Northwest Territories, respectively) were characterized by a short night length for hardiness induction, the 42 °N ecotype (Utah A and B) by a long night length for hardiness induction, while the F₁ was intermediate to the parents. Results from reciprocal crosses indicated there was no significant unilateral maternal influence on cold acclimation. Acclimation responses of the F₂ were highly variable but generally ranged between the parental extremes. However, three individuals from the 42 ° × 62 °N crosses exhibited greater cold resistance than the northern parent on two successive freezing test dates. F₂ plants were also found with less freezing resistance than the southern parent. Backcrosses to the southern parent produced progeny with acclimation patterns resembling that of the southern parent and were significantly less hardy than the F₂ in early freezing tests.Scientific Journal Series Paper No. 12,075 of the Minnesota Agricultural Experiment Station     

Changes in sugar content and fatty acid composition of in vitro sugar beet shoots after cold acclimation: influence on survival after cryopreservation

To cryopreserve sugar beet shoot tips using an encapsulation-dehydration technique, cold hardening of in vitro plants was needed to obtain high survival rates after freezing. Cold acclimation not only enhanced dehydration and freezing tolerance, but also induced several changes in sugar beet shoots. Plants contained greater amounts of sucrose, D-glucose and D-fructose and the fatty acid composition of lipids changed. Furthermore, the unsaturation level of membrane lipids, estimated by the (C_18:2 + C_18:1)/C_16:0 ratio, increased after cold hardening. These changes were correlated with better survival rates after cryopreservation.     

Feedback-limited photosynthesis and regulation of sucrose-starch accumulation during cold acclimation and low-temperature stress in a spring and winter wheat

Regulation of sucrose-starch accumulation and its effect on CO₂ gas exchange and electron transport were studied in low-temperature-stressed and cold-acclimated spring (Katepwa) and winter (Monopol) cultivars of wheat (Triticum aestivum L.). Low-temperature stress of either the spring or winter cultivar was associated with feedback-limited photosynthesis as indicated by a 50–60% reduction in CO₂ assimilation rates, twofold lower ATP/ADP ratio, and threefold lower electron transport rate than 20°C-grown control plants. However, no limitations were evident at the level of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) in low-temperature-stressed plants. Cold acclimation of the spring cultivar resulted in similar feedback-limited photosynthesis observed during low-temperature stress. In contrast, cold acclimation of the winter cultivar resulted in an adjustment of CO₂ assimilation rates to that of control plants. However, we show, for the first time, that this capacity to adjust CO₂ assimilation still appeared to be associated with limited triose phosphate utilisation, a twofold lower ATP/ADP ratio, a reduction in electron transport rates but no restriction at the level of Rubisco compared to controls grown at 20°C. Thus, contrary to previous suggestions, we conclude that cold-acclimated Monopol appears to exhibit feedback limitations at the level of electron transport characteristic of cold-stressed plants despite the maintenance of high rates of CO₂ assimilation. Furthermore, the differential capacity of the winter cultivar to adjust CO₂ assimilation rates was associated with higher levels of sucrose accumulation and a threefold higher sucrose-phosphate synthase activity despite an apparent limitation in triose phosphate utilisation.Abbreviations AGPase ADP-glucose pyrophosphorylase - FBPase fructose-1,6-bisphosphatase - Fru 6-P fructose 6-phosphate - Fru 1,6-BP fructose 1,6-bisphosphate - Glc 6-P glucose 6-phosphate - PGA 3-phosphoglyceric acid - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose 1,5-bisphosphate - SPS sucrose-phosphate synthase - Triose-P triose phosphate     

ABA and gibberellin-like substances during prehardening,cold acclimation,de- and reacclimation of oilseed rape

Previously published results showed that high relative reduction state of PSII (PSII excitation pressure) during both early seedling growth (prehardening) as well as cold deacclimation caused significant changes in growth pattern. The differences in elongation growth rate were related to the cold acclimation of photosynthetic apparatus and to frost resistance. To study changes in the hormonal balance connected with alterations in elongation growth rate observed during prehardening and deacclimation under different PSII excitation pressure (modulated by day-temperatures), endogenous concentration of ABA, GA₃ and GA-like substances (GAs) were analysed. Analyses were also performed during cold acclimation and reacclimation of plants characterized by different elongation growth rate triggered by prehardening or deacclimation under different day-temperatures. Growth under high PSII excitation pressure (prehardening) resulted in a significant increase in ABA and a considerable decrease in GAs contents. On the other hand, different ABA content played almost no role in controlling growth rate during cold deacclimation and subsequent reacclimation, when the induction of elongation growth was connected with the changes in concentration of GAs including GA₃. The possible role of ABA and GAs in controlling prehardening, cold acclimation and deacclimation is discussed.     

Genotypic differences in cold tolerance are masked by high sucrose and cytokinin in shoot cultures of sugarbeet

Cold tolerance of field grown plants and shoot cultures of a commercial sugarbeet cultivar, Hilma, was compared with that of two cultivars bred for improved cold tolerance, Monofeb and Winter Hybrid 88619. Leaves of Monofeb and Winter Hybrid 88619 showed an increase in frost tolerance compared to Hilma, as assessed by electrolyte leakage measurements, in both July, and November. However, all varieties exhibited acclimation in the latter month. Similar qualitative differences between cultivars were detected in shoot cultures only when maintained on low (1%) sucrose medium, without added plant growth regulators. The use of high (3%) sucrose and benzyladenine, which releases apical dominance producing multiple shoots, each contributed to a substantial lowering of the temperature at which cold-induced damage occurred in leaves. Under these conditions varietal differences were masked. The implications of these findings in regard to in vitro selection for improved cold tolerance in organized cultures are discussed.Abbreviations BA benzyladenine - MS Murashige & Skoog (1962)     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

In vitro shoot culture of wild Oryzae and other grass species

A method was developed for the in vitro clonal propagation of shoots from a range of wild rice and other grass species that have important genetic traits such as drought resistance and salinity tolerance. The axenic multiple shoot cultures, which were suitable for DNA and protein extraction or direct protoplast isolation, could be maintained without subculture for between 2 and 3 months or rapidly multiplied for the subsequent production of mature plants and seeds. Basal sections of the micropropagated shoots also provided novel explants for the production of highly embryogenic callus, from some species, that could be regenerated into green plants. It is envisaged that this clonal propagation technique could aid the genetic manipulation of cultivated rice by providing a means to vegetatively conserve valuable genetic resources, a technique to rapidly multiply novel hybrid material and a source of embryogenic callus that will allow the application of biotechnological techniques, such as somatic hybridization and genetic transformation, to previously unexploited species.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - PAR photosynthetically-active radiation     

Variability in storage potential of banana shoot cultures under medium term storage conditions

Shoot cultures of 401 banana clones were conserved under slow growth conditions (16±1°C, 25mol m^–2 s^–1). Storage duration-defined as 60% survival time of 20 shoot cultures of a clone-averaged 334 days. However, large differences occurred among the different genomic (sub)groups and even within the same (sub)group. East-African highland bananas and non-plantain AAB bananas can be stored for significantly longer periods. Shoot tip cultures of another 41 banana clones conserved at higher ambient temperature (22±3°C) needed to be subcultured sooner (every 220 days on average).Abbreviations BA 6-benzyladenine - CIRAD Centre de Coopération Internationale en Recherche Agronomique pour le Développement - IAA indole-3-acetic acid - IBPGR International Board for Plant Genetic Resources - INIBAP International Network for the Improvement of Banana and Plantain - PPF photosynthetic photon flux - QDPI Queensland Department of Primary Industries     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

The influence of cold acclimation on antioxidative enzymes and antioxidants in sensitive and tolerant barley cultivars

In order to better understand the role of cold acclimation in alleviating freezing injury, two barley cultivars with different cold tolerance, i.e. a sensitive cv. Chumai 1 and a tolerant cv. Mo 103, were used. The freezing treatment increased leaf soluble protein content more in the tolerant cultivar than in the sensitive one. Cold acclimation increased H₂O₂ content of the two cultivars during freezing treatment, especially in Mo 103. Glutathione and ascorbate contents during freezing and recovery were significantly higher in cold-acclimated plants than in non-acclimated ones. Activities of peroxidase, ascorbate peroxidase and glutathione reductase were also higher in cold-acclimated plants than non-acclimated plants during freezing treatment. However, there was no significant difference between cold-acclimated plants and the control plants in catalase activity. It may be assumed that cold acclimation induced H₂O₂ production, which in turn enhanced activities of antioxidative enzymes and synthesis of antioxidants, resulting in alleviation of oxidative stress caused by freezing.     

Induction of root and shoot formation from root meristems ofPetunia hybrida

Summary A procedure has been developed for the induction of root or shoot formation from root meristems of germinated seeds ofPetunia hybrida. Root formation was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine (BA) (0–0.5 mg/l) and naphtaleneacetic acid (NAA) (0.05–2.0 mg/l). Induction of predominantly shoot formation was obtained on MS medium containing the following combinations of hormones (in mg/l): 0.05–0.5 NAA and 0.25–2.0 BA. Complete plant formation was obtained after rooting of the shoots on MS medium supplemented with IAA (0–2.0 mg/l) or NAA (0-0.5 mg/l).     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.