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1.
Summary Nodal segments from in vitro cultured Petunia hybrida were grown under varying cultural conditions. The origin of nodal explants influenced vitrification. Basal segments formed
a higher percentage of vitreous shoots than did the upper nodes. A method was developed for including polyethylene glycol
with Gelrite to obtain gelled media of varying water potentials. Media water potential from −0.31 to −1.2 MPa had no effect
on controlling the level of vitrification. Gelrite promoted vitrification but GIBCO agar, alone or in combination with Gelrite,
reduced its occurrence. Lowering media NH 4 content reduced vitrification, whereas sealing culture vessels with parafilm increased it. As it is now possible to control
normal and vitreous plant development in Petunia, this can be used as a model system for studying the physiology and biochemistry of this developmental abnormality. 相似文献
2.
Summary Tissue cultures on properly solidified Gelrite media generally showed superior shoot proliferation and rooting, as well as
shoot and root vigor and callus development to those on TC agar. Vitrification, or hyperhydricity, was observed in both Gelrite
and agar media and minimized by increasing the gel concentrations. Rigidity of Gelrite media depended on combined levels of
MS macrosalts, basal nutrient formulations, sucrose concentration, pH, and Gelrite concentration. Most MS macrosalts increased
hardness of Gelrite gels; NH 4NO 3 had a decreasing effect. Rigidity of TC agar gels increased with reductions of MS macrosalts. A slightly softer Gelrite medium
resulted when sucrose was excluded. Both Gelrite and agar media were softer at lower pHs and harder at higher pHs. Activated
charcoal and mannitol increased gel hardness, and more noticeably of agar gels. NaCl addenda reduced rigidity, with their
effects being more pronounced on Gelrite than on agar gels.
Gelrite is a trademark of Kelco Division of Merck & Co. (San Diego, CA), manufacturer of the gellan gum. Phytagel is a
trademark of Sigma Chemical Co. (St. Louis, MO) for repackaged Gelrite. TC agar used for comparisons in this investigation
is plant tissue culture tested agar obtained from JRH Biosciences (Lenexa, KS). 相似文献
3.
Summary Enzymatically isolated leaf-derived protoplasts of peppermint ( Mentha piperita L.) were cultured in modified B5 medium containing 1 mg/l NAA, 0.4 mg/l BA, 0.5% sucrose, 0.5 M mannitol and 0.1% Gelrite (first medium). After 30 d culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. Gelrite medium blocks were transferred into liquid medium to promote further growth. Colonies of 0.5 mm transferred to 0.2% Gelrite solidified medium (same components as first medium) formed green calli (1–2 mm) under incubation in the light. Green calli transferred to differentiation medium (B5, 0.1 mg/l NAA, 5 mg/l BA, 2% sucrose, 0.2 M mannitol, 0.2% Gelrite) developed shoot buds after 3–4 weeks. Whole plants were recovered following rooting of shoots in B5 medium without hormones.Abbreviations BA
6-benzylaminopurine
- NAA
-naphthaleneacetic acid
- KIN
kinetin
- ZEA
zeatin
- CPW
cell and protoplast wash solution
- B5
Gamborg et al. (1968) mineral elements
- MS
Murashige and Skoog (1962) mineral elements 相似文献
4.
Shoot tips of York and Vermont Spur Delicious apples ( Malus domestica Borkh.) were cultured in vitro to test the influence of K +, Mg ++ and gelling agent concentrations on vitrification. These concentrations were 20.05, 14.05 and 8.05 mM K +, 1.5 and 3.0 mM Mg ++, 7.0 g/l Difco Bacto agar and 1.0, 1.5 and 2.0 g/l Gelrite. The lowest K + level produced a higher percentage of vitrified shoots, affected tissue appearance, reduced shoot number and shoot elongation and apparently altered shoot metabolic activity. Gelrite consistently produced vitrified leaves and stems, even though media gelled with 1.5 g/l Gelrite presented the same apparent gel firmness as using 7 g/l Difco Bacto agar, which did not induce vitrification. Less shoot elongation, fewer total shoots, and more usable shoots of York were obtained on Bacto-agar, while similar but less noticeable effects were obtained with Vermont Spur Delicious. The results presented here show that vitrification can be studied in a standardized system in which the only change is substitution of one gelling agent for another. 相似文献
5.
The effects of xylooligosaccharides isolated from the cell walls of Betula platyphylla var. japonica on cells and protoplasts
of Pinus radiata were examined. The addition of a semi-purified mixture of xylooligosaccharides at a concentration of 5μg.ml −1 promoted elongation of cultured cells, whereas the neutral fraction of this mixture had no effect; a similar effect was seen
in the presence of conditioned medium. The unfractionated mixture of xylooligosaccharides was also found to enhance the viability
of protoplasts prepared from cell cultures of Pinus radiata in a concentration dependent manner, highly similar to the effect
provided by addition of medium conditioned by pine cells. Such effects are considered to be due to the addition of components
that play a structural role in the cell wall of pines. It is inferred that the acidic components of the xylooligosaccharide
mixture derived from t Betula are responsible for this effect in the distant pine species. It is speculated that acidic xylooligosaccharides
operate either by replacing, or mimicking, the natural cell wall components required for growth and development of pine cultured
cells.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
6.
Observations on the effects of different degrees of rigidity of both an agar (Tayio) and a non-agar (Gelrite) gel on the uptake of radiolabelled N 6-benzyladenine ( 14C-BA) were also extended to mode of application and positioning of the explant. Regression analysis showed a highly significant inverse correlation between 14C-BA accumulation and degree of gel stiffness. Significantly greater numbers of adventitious buds per explant were induced at low to medium levels of rigidity (2.5–10 g Tayio 1 −1, 1–5 g Gelrite 1 −1); this advantage was almost completely nullified at the lower levels (2.5 and 5.0 g Tayio 1 −1, 1 and 1.5 g Gelrite 1 −1) as a result of the high incidence of vitrification. In addition to turgor distension, vitrified buds displayed cellular damage. Explants with their cotyledons flattened onto the agar surface accumulated less 14C-BA after 96 h than upright explants, but produced greater numbers of adventitious buds, pseudobuds and phylloids. It was suggested that BA was taken up only by "target" cells, presumably the differentiating subsidiary cells of those stomatal complexes in surface contact with the medium. Pulse treatments of relatively short durations (2 h) with optimal concentrations of BA (ca 125 μ M ), followed by subculturing on hormone-free media gelled with 10 g agar 1 −1, produced a satisfactory balance between yield and competence of adventitiously-induced buds. 相似文献
7.
Corydalis ambigua, a perennial herb of the family Papaveraceae, was micropropagated through somatic embryogenesis starting from sliced tubers. Somatic embryos were proliferated on Linsmaier and Skoog medium of a half strength containing 2% sucrose and 0.1 M indole-3-acetic acid or indole-3-butyric acid solidified with 0.2% Gelrite. Somatic embryos were germinated and grew on plant growth regulator-free White's medium supplemented with 2% sucrose and 0.8% agar in the dark at 0–4°C for 6 months to give rise to microtubers that could be potted out of culture tubes. Three strains of micropropagated plants cultivated outdoors for 5 years gave different tetrahydroprotoberberine-type alkaloids pattern, respectively. The variation of tetrahydroprotoberberine-type alkaloid (corybulbine, corydaline and cavidine) content within a strain was not significantly different from that of the corresponding alkaloid in the wild plants. 相似文献
8.
Summary Although pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations of this crop have been focused on propagation. The procedure involving the use
of temporary immersion bioreactors is one of the most outstanding because of its high multiplication rate. We previously recorded
specific protease activity in the culture medium during the pre-elongation step of this protocol. Therefore, we decided to
modify the culture medium composition of this phase looking for an increase in protease excretion. Four independent experiments
were performed to evaluate the effects of different levels of sucrose (0–350.4 m M), inorganic salts [0–200% Murashige and Skoog (MS) salt strength], inositol (0–2.20 m M), and thiamine (0–1.2μ M). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity,
and specific protease activity in culture media. Specific protease activity, the most important indicator recorded, was highest
with 262.8 m M sucrose, 100% MS salt strength, 0.3 μ M thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 m M sucrose, 100% MS salt strength, 0.55 m M inositol, 0.3 μ M thiamine) are not always adequate for protease excretion. 相似文献
9.
There has been a lack of research into equine sperm vitrification to date, but studies of other species suggest it may have significant potential. To evaluate the impact of various cryoprotectant agents (CPA) and vitrification on equine sperm quality, a controlled study was carried out. A total of 12 ejaculates were subjected to exposure to CPA and vitrification. Sperm was diluted in a range of CPA: fresh, control (BSA), sucrose (0.15M, 0.3M and 0.5M), trehalose (0.15M, 0.3M and 0.5M) and the combination of sucrose and trehalose (M1: 0.15M sucrose+0.5M trehalose; M2: 0.5M sucrose+0.15M trehalose). Sperm motility, viability, acrosome integrity and DNA fragmentation were assessed at the time of CPA exposure and after vitrification. The exposure of spermatozoa to various concentrations of sucrose and/or trehalose significantly reduced sperm motility, with lower concentrations resulting in higher sperm motility. Sperm viability and DNA fragmentation did not vary after exposure to CPA, but acrosome integrity fell significantly when spermatozoa were exposed to CPA with high osmolality. When spermatozoa were vitrified, motility values were significantly higher than those obtained during the exposure. Low concentrations of sucrose (0.15M and 0.3M) and trehalose (0.15M) showed the best progressive sperm motility. The vitrification-warmed procedure significantly reduced sperm viability and acrosome integrity, but DNA did not vary with any of CPA used. Equine sperm vitrification demonstrates a low capacity for preserving sperm motility, and extenders containing trehalose or sucrose at lower concentrations are associated with a better protective effect on sperm motility. After vitrification, acrosome and plasma membranes were severely impaired, while the DNA structure was maintained. Equine spermatozoa partially recover the motility after vitrification, but there is a need for further studies into the preservation of sperm membranes. 相似文献
10.
The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles ( p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose ( p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended. 相似文献
11.
Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration,
alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations
(0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification,
apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration,
treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid
nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification
and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant
re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day
sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel. 相似文献
12.
The effects of dietary inositol with sucrose stimulation on chewing and swallowing motor patterns in the larvae of Bombyx mori L. are investigated. Feeding activities of the larvae are significantly enhanced by a test diet containing an inositol–sucrose mixture compared with a test diet of sucrose only. Motor patterns of the mandibular closer muscle are accelerated with shorter burst durations and shorter inter‐burst intervals with the test diet of inositol–sucrose compared with sucrose. In terms of swallowing behaviours, inositol–sucrose shortens the duration of drinking. Motor patterns of the cibarial compressor muscle are accelerated with shorter burst durations and shorter inter‐burst intervals with the inositol–sucrose mixture. Peripheral interactions between inositol‐ and sucrose‐sensitive cells in the maxilla are not detected. Thus, such interactions cannot explain the positive effects of inositol on chewing and swallowing. Responses of inositol‐sensitive cells in the epipharyngeal sensillum are not affected by sucrose. These results suggest that dietary inositol can modify chewing and swallowing motor patterns when coupled with sucrose stimuli. These modifications may occur in the central neural networks involved in chewing and swallowing motor patterns but not in peripheral sensory interactions. 相似文献
13.
Teucrium polium L. with the common name of Felty Germander is one of the plants flora that is widely used in folk medicine in many Middle East countries, it is an endangered plant species and must be highly considered for preservation. Cryopreservation of T. polium by vitrification and encapsulation-dehydration was successfully achieved in this study. Shoot-tips were excised aseptically from in vitro grown plants and incubated for 3?days on solid hormone free-Murashige and Skoog (HF-MS) media supplemented with 0.3?M sucrose under complete darkness at 24?±?1?°C. In vitrification, shoot-tips were loaded in 0.4?M sucrose and 2?M glycerol for 20?min followed by desiccation with different combinations and concentrations of plant vittrification solution 2 (PVS2), before immersion in Liquid Nitrogen (LN). Whereas for the encapsulation-dehydration; shoot-tips were encapsulated in calcium alginate and dehydrated under laminar air flow cabinet for 0, 3, 6, or 9?h. A total of 60?% of the cryopreserved vitrified shoot-tips survived when desiccated in concentrated PVS2 solution for 20?min, whereas, 28?% of the cryopreserved vitrified shoot-tips were regrown after 20?min of desiccation by two step increase in PVS2 concentration. Complete survival were obtained for the non-cryopreserved encapsulated shoot-tips treated for 3?days in 0.5?M sucrose with MS media without or with 3?h of dehydration, whereas, only 20?% of the cryopreserved encapsulated shoot-tips were regrown. The procedures developed in this study are easy to handle and produced a high levels of shoot formation. 相似文献
14.
Myo -inositol is a precursor of many plant metabolites, including polyols, cell wall components and phosphoinositides. The first committed step in the de novo myo -inositol synthetic pathway is catalysed by the enzyme 1D- myo -inositol 3-phosphate synthase (MIPS; EC 5.5.1.4 ), which converts D-glucose 6-phosphate to 1D- myo -inositol 3-phosphate. Suppression of MIPS activity by an antisense RNA approach in transgenic potato ( Solanum tuberosum L.) plants to below 20% of the wild-type level in leaves resulted in strongly reduced levels of inositol, galactinol and raffinose (approximately 7%, 5% and 12%, respectively, of wild-type values). In contrast, increases were observed for concentrations of hexose phosphates (up to 1.7-fold), sucrose (twofold) and starch (two- to fourfold). Transgenic plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence and a decrease in overall tuber yield. These observations indicate a crucial role for myo -inositol in plant physiology and development. 相似文献
15.
A simple and efficient method for multiplication of vanilla ( Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters of plantlets, by cutting the remaining base of these plant clusters into segments of about 1 cm in length.
After 4 months of culture on multiplication medium, 100% of IFEs produced up to 15 new shoots per explant, providing an efficient
additional method for in vitro propagation of vanilla that maximizes the use of available material. Cryopreservation of apices from in vitro grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%)
of new shoots were obtained for apices derived from clusters of in vitro plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid
MS medium with 0.3 M sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to plant vitrification
solution PVS3 for 30 min at room temperature. Even though the cryogenic protocol needs to be optimized to improve results,
this work represents the first successful report of cryopreservation of vanilla apices. 相似文献
16.
Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher ( V. berlandieri × V. rupestris), 41B ( V. vinifera × V. berlandieri) and several table grape and wine cultivars ( Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l –1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species. 相似文献
17.
Levels of the major carbohydrates (hexoses, inositol, sucroseand starch) and activities of the enzymes (invertase, amylaseand starch phosphorylase) were assayed in the leaves and stemsof tomato plants. The transport system of the plants was simplifiedto one leaf and one associated truss either retained or removed.The experiment was carried out at two light intensities, 80and 20 W m 2. Truss removal immediately increased thelevels of hexoses and starch in leaves under high light, butthose of sucrose and inositol were relatively unaffected withina day. Three days after truss removal the levels of sucrose,hexose and starch had reached a maximum. Under low light, leafsugar levels were lower and did not increase following trussremoval. The stem appeared to act as a substitute sink in thoseplants where the truss had been removed. Invertase and amylase activities were not significantly affectedby the treatments, but loss of starch phosphorylase activityduring the experiment was prevented by truss removal. Tomato, source-sink relationships, carbon, carbohydrate 相似文献
18.
A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite
in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial
light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best
results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin
were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation
rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD
of 65 μmol m −2 s −1 and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m −2 s −1 and photoperiods ranged from 8–16 hours.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
20.
In this study, an effective root-based cryopreservation method was developed for Hypericum perforatum L., an important medicinal species, using in vitro plants. A systematic approach was applied to determine effective combinations of protocol steps such as preculture, osmoprotection, vitrification solution treatment, and unloading, followed by protocol optimization using a single-factor approach. The effects of root section type (root tips, middle sections, or basal sections), duration of root section culture after excision, and donor plant age were also investigated. In a wild genotype, middle and basal root sections excised from 8-wk-old plants and cryopreserved at the age of 10 d after excision showed the highest plant regrowth after cryopreservation. In the optimized protocol, root sections were precultured in 10% (w/v) sucrose for 17 h, osmoprotected with a solution composed of 17.5% (w/v) glycerol and 17.5% (w/v) sucrose for 20 min, followed by a vitrification solution of 40% (w/v) glycerol and 40% (w/v) sucrose for 30 min, and cryopreserved using aluminum foil strips (droplet-vitrification). After rewarming in preheated 25% (w/v) sucrose solution and 30-min unloading, root segments were recovered on medium supplemented with 1.0 mg L−1 gibberellic acid and showed 78% plant regrowth. This cryopreservation method was successfully adapted for five elite lines of H. perforatum with a 45 to 87% regrowth rate after cryopreservation. These results suggest that root cryopreservation may be an effective method for medicinal plant conservation and should be tested with a broader range of species. 相似文献
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