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1.
Summary The effects of different factors on the control and reversion of hyperhydricity during the in vitro propagation of Prunus armeniaca were studied. Treatments that decreased the hyperhydricity but did not affect micropropagation rates were the use of the
bottom cooling system for 1 or 2 wk and agargel as gelling agent in ‘Helena’, whereas the best results were obtained with
the bottom cooling system for 2 wk and the use of 0.8% agar as gelling agent in ‘Lorna’. Hyperhydric shoots reverted to normal
after keeping them for 3 wk in the bottom cooling system. 相似文献
2.
The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N 6-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene. 相似文献
3.
In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity
in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in
the induction of hyperhydricity. Shoots were grown on media with different NH 4
+ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N 6-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH 4
+, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added
to media containing low NH 4
+ (10.3 mM). However, in the presence of higher NH 4
+ concentrations, CKs increased hyperhydricity in a concentration-dependant manner, suggesting that they were capable of inducing
this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were
produced on media with low NH 4
+ and low BA or zeatin (5 μM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying
the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for
the conservation of this species. 相似文献
4.
Hyperhydricity is a physiological disorder during plant tissue culture that seriously affects regeneration and micropropagation. In this study, Dendrobium officinale plantlets were cultured on solid Murashige and Skoog (MS) medium supplemented with plant growth regulators and various concentrations of sucrose, agar, and polyethylene glycol (PEG)-6000 to explore the effect of osmotic stress on hyperhydricity. The results show that low concentrations of sucrose or agar, as well as PEG-6000 at various concentrations, significantly increase the hyperhydric rate of D. officinale, whereas high concentrations of sucrose or agar did not. Furthermore, high concentrations of PEG-6000 significantly increase total water content, free-water content, relative electrical conductivity, and peroxidase (POD) activity of D. officinale plantlets, whereas they significantly decrease bound-water content, proline content, soluble protein content, soluble sugar content, and superoxide dismutase (SOD) activity. These results indicate that PEG-6000 disrupts the antioxidant system and water metabolism in D. officinale plantlets, as well as increases cell membrane permeability, which might be the key factors for the occurrence of hyperhydricity in this species. 相似文献
5.
Hyperhydricity is a physiological disorder frequently affecting shoots propagated in vitro. Since it negatively affects shoot multiplication vigor, and impedes the successful transfer of micropropagated plants to
in vivo conditions, hyperhydricity is a major problem in plant tissue culture. In commercial plant micropropagation, there are reports
of up to 60% of cultured shoots or plantlets which demonstrate hyperhydricity, which reflects the pervasiveness of this problem.
The phenomenon has been correlated to water availability, microelements, and/or hormonal imbalance in the tissue culture.
In this study, the ultrastructure and the characteristics of reactive oxygen species between hyperhydric and normal shoots
of garlic were studied. We observed that in some cells of hyperhydric tissues, the intranuclear inclusion was separated, the
mitochondrion was swollen and its intracristae had splits, the organelles were compressed against the cell wall, and the chloroplasts
and intergranal thylakoids were also compressed. Additionally, the content of chlorophyll and soluble protein in hyperhydric
shoots decreased significantly. For instance, chlorophyll a decreased 43.61%, chlorophyll b decreased 49.29%, chlorophyll
a+b decreased 48.10%, and soluble protein dropped 47.36%. In contrast, the O 2 generation rate and H 2O 2 level increased 45.36% and 63.98%, respectively, obviously higher than the normal shoots. Lipoxygenase activity and malondialdehyde
content in the hyperhydric shoots increased significantly, while the electrolyte leakage rose, indicating a serious membrane
lipid peroxidatic reaction. Superoxide dismutase, peroxidase, catalase, glutathione peroxidase, and ascorbate peroxidase activities
in hyperhydric tissue were all significantly higher than in normal leaf tissue. The antioxidant metabolism demostrated a close
connection between hyperhydricity and reactivated oxygen species. 相似文献
6.
Addition of the growth retardant ancymidol to Narcissus shoots and lower inner leaf sections isolated from shoots cultured in liquid medium induced hyperhydric malformations associated
with morphogenetic changes. Meristematic centers initiated on the basal proximal ends appeared over the entire surface of
the hyperhydric leaf sections after 6 weeks in culture. The meristematic centers which formed clusters on the leaf sections
developed later into buds. In leaf sections grown in the liquid medium lacking ancymidol, hyperhydricity was not induced,
and regeneration was not observed. Starch and protein levels and ascorbate peroxidase and catalase activities were examined
in shoots and isolated leaf sections that were either hyperhydric or non-hyperhydric. In ancymidol-treated, hyperhydric leaf
sections, ascorbate peroxidase and catalase activities were lower than in control, untreated leaf sections. The changes in
starch and protein levels and in antioxidant enzymatic activities appeared to be related to the onset of meristematic-center
initiation and further bud development on Narcissus hyperhydric leaf sections.
Received: 6 May 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000 相似文献
7.
Carnation plantlets ( Dianthus caryophyllus L.) cultured in vitro often develop morphological and physiological anomalies, a phenomenon called hyperhydricity, which impairs their survival
ex vitro. When the agar concentration of the growth medium was increased (from 0 to 12 g dm −3), thereby reducing water availability, the hyperhydricity of those adventitious shoots regenerated from carnation petals
decreased. This was accompanied by a progressive fall in the water content of shoots (94.9 to 91.4 %), fresh mass (from 57.2
to 1.8 mg), number of leaf parenchyma cell layers (from 9.3 to 7.7), and the size of these cells (from 968 to 254 μm 2). However, the number of regenerated shoots also decreased (17.7 in 2 g dm −3 agar to 4.3 in 12 g dm −3). Similarly, in ventilated tubes, which exhibit a lower relative humidity than tightly closed tubes, shoot organogenesis
diminished up to 28 %, in tandem with shoot water content. Thus, relative humidity and water availability in culture vessels
do not only influence shoot hyperhydricity in carnations, but also greatly affect adventitious shoot organogenesis. 相似文献
8.
Summary A micropropagation protocol was developed for Cinnamomum camphora (L.) Sieb., using as initial explants 3–5-mm shoot tips from newly emerged laterals of 2-yr-old trees. Performance of small
shoot tips was compared with that of 2.0-cm nodal segments during subculture. Murashige and Skoog medium (MS) supplemented
with different concentrations of N 6-benzyladenine (BA) or thidiazuron (TDZ) was used to examine shoot proliferation. In separate experiments, MS was supplemented
with 1-naphthaleneacetic acid (NAA) for rooting of shoots, and the commercial preparation EM 2 for prevention of hyperhydricity. BA stimulated shoot formation and callus development, whereas TDZ promoted only callus
development. Both cytokinins induced hyperhydricity when small shoot tips were used, with severity being directly related
to concentrations. Hyperhydricity was avoided in subcultures by using larger nodal segments. EM 2 did not alter degree of hyperhydricity but suppressed callus development and strongly promoted shoot multiplication. The
number of new shoots after a 6-wk subculture was 9 per nodal segment when supplemented solely with 4.4 μ M BA and 18 per segment when further supplemented with 1000 mg EM 2 per I. Rooting of shoots occurred best when supplemented solely with 0.54 μ M NAA, averaging 7 roots per shoot in 4 wk. Ninety percent of rooted shoots survived transfer to the greenhouse. 相似文献
9.
Hyperhydricity symptoms are common and significant during the in vitro culture of Dianthus chinensis L. and greatly affect the micropropagation and regeneration of cultured plantlets. However, effective measures for preventing such abnormalities have not been developed for this species. Silver nitrate (AgNO 3) has been shown to revert hyperhydric plantlets to a normal state. Nevertheless, the effect of AgNO 3 on the prevention of hyperhydricity and the underlying mechanisms remain unclear. In the present study, 98.7% of the Dianthus chinensis L. plantlets cultured in a hyperhydricity induction medium (HIM) developed symptoms of hyperhydricity; however, hyperhydricity symptoms were inhibited to different degrees when D. chinensis L. plantlets were cultured in HIM supplemented with various concentrations of AgNO 3. In particular, approximately 97% of the D. chinensis L. plantlets grew normally and did not show any symptoms of hyperhydricity when cultured in HIM supplemented with 30 μmol L ?1 AgNO 3. Compared with the plantlets cultured in HIM alone, the plantlets cultured in HIM containing AgNO 3 displayed dramatic decreases in water content, ethylene content, and reactive oxygen species (ROS) production (particularly regarding H 2O 2 accumulation in guard cells) and showed increased antioxidant enzyme activity, stoma aperture, and water loss. These changes not only prevented excess water from accumulating in the tissues of plantlets but also improved the antioxidant capacity of plantlets, ultimately resulting in the prevention of hyperhydricity. 相似文献
10.
This study was made to clarify the effects of cytokinin type and their concentrations on shoot proliferation and hyperhydricity in in vitro Pyrus pyrifolia N. (`Hosui' and `Kosui') shoots. The shoots were subcultured in a woody plant medium supplemented with 0.5 M 3-indolyl-butyric acid, 3% (w/v) sorbitol, 0.8% (w/v) agar, and with cytokinins kinetin, 6-benzylaminopurine (BA), N-(2-chloro-4-pyridyl)- N9-phenylurea (CPPU), 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) added at concentrations 0.44, 4.40, 11.0 and 44.0 M. The highest number of shoots was induced by adding BA at concentration 11.0 M, then by 4.4 M BA, in both cultivars. TDZ and CPPU caused greater hyperhydricity in cultured explants than BA and kinetin. `Kosui' was more susceptible to hyperhydricity compared with `Hosui'. 相似文献
11.
In vitro-grown shoots and calli of Withania somnifera, an important medicinal plant, were exposed to various types of salts under in vitro culture conditions. Membrane permeability, lipid peroxidation, and the antioxidant system increased in shoots as well as in unorganized callus tissues under all the three concentrations of KCl, NaCl, KNO 3, NaNO 3, and CaCl 2. The growth responses of shoots and callus cultures under various salt treatments revealed that the tissue could grow better under NaCl and KNO 3 compared to other salts and the in vitro shoots appeared healthy at 50?mM concentration of NaCl and KNO 3. The activity of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, lipoxygenase, polyphenol oxidase, and glutathione reductase increased under salt treatments, especially at higher concentrations. The greatest activity increase was recorded for peroxidases, whereas CAT was the least responsive. Only two isoforms, Mn-superoxide dismutase (Mn-SOD) and Fe-SOD, could be visualized in callus tissue while Cu/Zn-SOD was absent. Diaphorase 4 was totally missing in callus tissue and was detected only in shoots. Phenolics accumulated at all the concentrations of the salts tested as an induced protective response. The higher concentration of CaCl 2 produced maximum increases in antioxidants and enzymatic activities compared to other salts. Thus, for W. somnifera the presence of excess calcium in the growing medium is most deleterious compared to other salts. Results also suggest that the nonenzymatic and enzymatic antioxidant systems of both the tissues played a primary role in combating the imposed salt stress. 相似文献
12.
In vitro propagated plants of Mammillaria
gracilis Pfeiff. ( Cactaceae) develop calli without any exogenous growth regulators. This habituated tissue spontaneously regenerates morphologically
normal as well as hyperhydric shoots. In this study, a possible involvement of activated oxygen metabolism in habituation
and hyperhydricity in in vitro propagated plants of Mammillaria
gracilis Pfeiff. ( Cactaceae) was investigated. Significantly higher malondialdehyde (MDA) and carbonyl contents as well as hydrogen peroxide (H 2O 2) production were observed in habituated callus (HC), hyperhydric regenerated shoots (HS), and tumors (TT) in comparison to
normal regenerated shoots (NS). Lipoxygenase (LOX) activity showed a similar trend, with a clear increase in activity in HC
and HS. The activities of antioxidative enzymes, namely, peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT),
were also higher in HC, HS, and TT, whereas an increase in superoxide dismutase (SOD) activity was observed in HC and HS.
The majority of antioxidative isoenzymes were common to all cactus tissues, although a few tissue-specific bands were noticed.
Significant decreases in phenylalanine ammonia lyase (PAL) activity, total phenolic content, and lignification were found
in HS, HC, and TT in comparison to NS. Our results showed the appearance of a prominent oxidative stress in HC, HS, and TT
as well as a strong induction of the antioxidant system indicating that activated oxygen metabolism could be involved in habituation
and hyperhydricity and linked to the loss of tissue organization in M. gracilis.
B. Balen and M. Tkalec contributed equally to this work. 相似文献
13.
Hyperhydric disorders occur frequently in plant tissues cultured in vitro and cause several morphological and physiological abnormalities. However, a systematic defense response is triggered by hyperhydric conditions. The accumulation of reactive oxygen species (ROS), activities of antioxidant enzymes and their immunoblots, and the proteome-level changes in normal versus hyperhydric shoots of carnation ( Dianthus caryophyllus) cultured in vitro were investigated. Total proteins were also extracted from the shoot and analyzed by two-dimensional electrophoresis. Among a total of 700 spots detected, only 40 had significant changes in abundance in the hyperhydric compared to the normal shoots, which were further identified by a mass spectrometer (MALDI-TOF MS). Most of them were involved in photosynthesis, RNA processing, and general metabolisms, while the rest were involved in secondary metabolic processes. These identified proteins in carnation shoots may provide novel evidences for stress tolerance against hyperhydricity. 相似文献
14.
Summary The type of gelling agent can influence to a large extent clonal propagation of Ranunculus asiaticus L. through axillary bud stimulation. In preliminary experiments we identified three agar brands (Oxoid=OX, Merck=MK, and
Roth=RT) which affect the availability of water and minerals to tissues in different ways. In the present study we investigate
the influence of these agars on the in vitro performance of Ranunculus. On OX and MK gels, growth was satisfactory, although the former had a more promotive effect on fresh and dry weight production
and on multiplication rate. Growth and development of shoots were poor on RT; shoot clumps showed symptoms of hyperhydricity,
with shoots having large dark-green malformed leaves and very elongated petioles. Epidermal strips of leaves from shoots grown
on the different gels and collected at the end of the culture period revealed differences according to the agar brand on which
the plantlets were cultured. Severe structural deformations of stomata could be detected on RT-grown shoots. The analyses
of the sugar content of the gel at the end of the culture period demonstrated that the explants grown on RT gels are strictly
dependent on the carbohydrates in the medium. On OX and MK gels the heterotrophic metabolism was lowered compared to RT-grown
explants. The agar brand on which plantlets were grown also influenced water retention capacity and water content of the shoots.
Experiments with tritiated water were undertaken to better understand the water fluxes inside the vessel and to investigate
the difference in “pump function” exerted by shoots cultured on the three gels. Shoots grown on OX media showed the best “pump
function,” which would account for the better results obtained on this gel. On the basis of the relationship between gel properties
and the growth of Ranunculus shoots, we conclude that the different physiological responses on the three gels are a reflection of different water and
nutrient availability in the different media. 相似文献
15.
A low cost micropropagation protocol to produce high quality root systems which are easy and economical to acclimatize is
essential for large-scale micropropagation of papaya ( Carica papaya L.). In this study, individual shoots (>0.5 cm) with 2 ∼3 leaves from in vitro papaya multiple shoots were cultured on MS agar medium containing 2.5 μM IBA under dark conditions for 1 week for root induction.
They were then transferred to agar or vermiculite media, containing half strength MS medium, under aerated or non-aerated
conditions, for root development. Rooting percentage of shoots cultured for 2 weeks in aerated vermiculite was 94.5%, compared
with 90.0% in non-aerated vermiculite, 71.1% in aerated agar, and 62.2% in non-aerated agar. Shoots with roots were acclimated
in vermiculite under 100% RH for 1 week and then under ambient conditions for 2 weeks in a temperature-controlled growth chamber
(28 °C). The survival rates of the plantlets were 94.5% from aerated vermiculite, 87.8% from non-aerated vermiculite, 42.2%
from aerated agar, and 35.6% from non-aerated agar. Thus, root induction in low-concentration IBA agar medium followed by
root development in vermiculite containing half strength MS medium under aerated conditions results in efficient rooting of
in vitro papaya shoots.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
16.
Adventitious shoots induced from maca calli on induction media without rare earth elements (REE) had higher water content
and lower soluble protein concentration when compared with shoots sprouted from maca seeds. Due to lower activities of antioxidative
enzymes, there were higher concentrations of H 2O 2 and malonyldialdehyde (MDA) in adventitious shoots than those in seed shoots. When La 3+, Ce 3+ and Nd 3+ (0.04 mM to 0.1 mM) were added to induction media, induction rates of the adventitious shoots were only affected slightly,
but hyperhydricity rates were significantly reduced. La 3+, Ce 3+ or Nd 3+ enhanced the activities of antioxidative enzymes in adventitious shoots, e.g. peroxidase (POD), catalase (CAT), ascorbate
peroxidase (APX), superoxide dismutase (SOD), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR). When
the concentrations of La 3+, Ce 3+ and Nd 3+ were 0.1 mM, the oxygen stress in adventitious shoots was decreased to levels similar to seed shoots, where most adventitious
shoots grew normally. 相似文献
17.
An axillary proliferating clone of Prunus avium L. was subcul- tured every four weeks on solid MS medium with agar as the gelling agent. Vitrification (hyperhydricity) of shoots was induced in one four week cycle with the same medium except that agar was replaced by gel- rite. During culture on the vitrifying medium, the water content of the shoots progressively increased with a parallel decrease in chlorophyll content. Cytological differences between the leaves and stems of the vitrified and normal shoots were detected by light and electron (both transmission and scanning) microscopy. Leaves of vitrified shoots were characterized by lower number of chloroplasts in the palisade parenchyma and by a defective cuticle. The stems of vitrified shoots had a less developed and lignified xylem tissue, lacked sclerenchy- matic areas and showed hypertrophy of the cortical parenchyma. More intense vacuolar activity with evagina- tions of the chloroplast envelope into the vacuole was noted in cells of vitrified leaves. 相似文献
18.
Shoots of the marubakaido apple rootstock grown in culture medium containing BBL agar presented significantly lower multiplication
rate (MR) compared to MRs found for shoots grown in medium containing A-7002, A-7921, Select, and Phytagar as gelling agents.
In addition, significant hyperhydricity was found for shoots grown in Phytagar and A-7921 agar-containing media. Analysis
of elastic (G′) and viscous (G″) modulus showed that for all of the five agar brands used in this study, G′ was always much
higher, i.e., typically one order of magnitude higher than G″, which characterizes a strong gel. G′ changed randomly with
time for all of the agar brands studied, except for BBL, which presented progressive decline in G′ throughout the culture
cycle. Examination of G′, within the same week, showed that Select agar always had the smallest G′, while Phytagar always
had the highest G′. Analysis of the loss tangent (tan δ = G″/G′), a better indicator for gel behavior compared to G′ isolated, showed that tan δ for Select and Phytagar were always
between tan δ values found for A-7002 and BBL. In addition, analysis of tan δ also indicated that BBL and Select agars showed a significantly weaker gel network, compared to Phytagar, A-7002 and A-7921
agars after the third week of culture. When seen together, these results indicate that shoot performance for the marubakaido
rootstock is not related to agar gel strength. In addition, the high hyperhydricity rate found for shoots grown on agars A-7921
and Phytagar could not be related to agar gel strength, as well. Analysis of HPSEC profiles indicated that the best performance,
i.e., multiplication rate, of marubakaido shoots in agars A-7002 and A-7921 is likely to be related to their lower polydispersity
and/or smaller amount of high molecular weight fractions, compared to BBL, Phytagar, and Select agars. 相似文献
19.
Summary Cultures of Minthostachys mollis were established from nodal explants obtained under aseptic conditions. Explants were cultured on Murashige and Skoog (MS)
half-strength medium containing 6-benzyladenine (BA), and/or naphthaleneacetic acid (NAA). Optimum numbers of healthy shoots
were induced on media containing 0.05 μ M NAA plus 2.2 μ M BA; higher concentrations caused more hyperhydricity and less extension. Rooting was achieved on half-strength MS medium
with 0.05 μ M NAA. Plantlets were acclimatized and successfully transferred to soil. Essential oil composition of the regenerated plants
were determined by gas chromatography/mass spectrometry and little differences were found in the essential oil composition
with the plant grown in the wild. 相似文献
20.
Avocado shoots were multiplied in vitro in two culture media of different consistency, double phase and solid medium, at three different irradiance levels: 35, 60
and 85 μmol m −2 s −1. Effects of culture and environmental conditions in multiplication rate, rooting capacity, hyperhydricity and leaf surface
morphology of microcuttings were evaluated. Double phase medium induced hyperhydricity, producing leaf microcuttings with
deformed stomata and low crystalline epicuticular waxes; microcuttings also showed reduced rooting capacity. By contrast,
solid medium promoted leaf area development on microcuttings and decreased hyperhydricity. Stomatal index was not affected
by these treatments but stomatal density was, interacting with the amount of irradiance applied. Increasing irradiance decreased
concentration of chlorophyll a and carotenoids in the leaf but did not affect leaf hyperhydricity.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
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