Effect of culture media and irradiance level on growth and morphology of Persea americana Mill microcuttings

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.     

Influence of gelling agent and cytokinins on the control of hyperhydricity in <Emphasis Type="Italic">Aloe polyphylla</Emphasis>

Shoot regeneration and occurrence of hyperhydricity in Aloe polyphylla were greatly affected by the type of gelling agent. The use of gelrite resulted in a significantly lower multiplication and almost four times higher hyperhydricity (65%) compared to agar-solidified medium. Gelrite was further selected to evaluate if hyperhydricity can be overcome by altering the physical properties of the gel, as represented by increasing gelrite concentrations. Four concentrations of gelrite (0, 2.4, 6 and 16 g l⁻¹) were tested in combination with zeatin, N⁶-benzyladenine (BA) or thidiazuron (TDZ). Almost all explants grown in liquid media in the presence of cytokinins became hyperhydric and lost their ability to regenerate. The greatest shoot formation was obtained on media with 2.4 g l⁻¹ gelrite and 5 μM zeatin or BA, however hyperhydricity was very high. Satisfactory reduction in hyperhydricity was achieved only at 16 g l⁻¹ gelrite, under which conditions the multiplication also decreased. The use of TDZ resulted in very low shoot regeneration and high hyperhydricity irrespective of the gelrite concentration.     

Control of hyperhydricity in micropropagated apricot cultivars

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.     

Reduction of hyperhydricity in tissue cultures of oregano (Origanum vulgare) by extracellular polysaccharide isolated from Pseudomonas spp

Hyperhydricity or vitrification is a physiological malformation affecting tissue culture-based propagation of several plant species. A Pseudomonas spp-mediated approach was recently developed to control hyperhydricity in oregano. This bacterium-induced prevention of hyperhydricity helped the establishment of clonal plants in the greenhouse without extensive acclimatization. The prevention of hyperhydricity was specifically linked to mucoid Pseudomonas spp and was characterized by high chlorophyll and reduced water content in oregano shoots. The focus of research reported in this paper was to purify the extracellular mucoid component from Pseudomonas spp and evaluate the effect on hyperhydricity in oregano tissue culture. The extracellular mucoid component was purified by ethanol precipitation. This extracellular mucoid component was confirmed to be a polysaccharide using gas chromatography-mass spectrometry. The effect of purified polysaccharide to prevent or reduce hyperhydricity was tested in oregano clone 0–1. The polysaccharide prevented hyperhydricity in oregano with reduced efficiency compared to bacterial inoculation. This was characterized by higher chlorophyll and reduced water content when compared to uninoculated/untreated oregano shoots. This confirms that the Pseudomonas spp-mediated hyperhydricity reduction in oregano is partially due to its extracellular polysaccharide. This provides a novel approach to develop a media formulation to control hyperhydricity in wide number of plant species where tissue culture is used for clonal propagation.     

Agar and ammonium nitrate influence hyperhydricity,tissue nitrate and total nitrogen content of serviceberry (Amelanchier arborea) shoots in vitro

Shoot tip cultures of Amelanchier arborea Michx.f. were grown on Murashige & Skoog or Woody Plant (WP) medium containing 4.4 M benzyladenine and various concentrations of agar. Increases in agar concentration affected various culture growth variables, decreased culture hyperhydricity and increased tissue nitrate concentration. Additions of ammonium nitrate to cultures grown on WP medium containing 0.4% agar increased all growth variables measured except percent dry weight. Hyperhydricity and tissue nitrate concentration also increase in response to increasing ammonium nitrate in the medium. Since hyperhydricity was shown to be both positively and negatively correlated with increases in tissue nitrate content, it is unlikely that tissue nitrate level alone directly affects hyperhydricity.Abbreviations BA benzyladenine - MS Murashige & Skoog - WP Woody Plant     

Prevention of hyperhydricity in micropropagated carnation shoots by bottom cooling: implications of oxidative stress

Carnation shoot cultures were micropropagated in two different agar concentrations (0.58 and 0.85%) and placed in a bottom cooling system or control conditions. During the culture period of 28 days, it was observed that relative humidity, hyperhydricity, dry weight, multiplication rate, and the activity of the antioxidant enzymatic system changed in relation to the agar concentration used and the application of bottom cooling. The percentage of hyperhydric shoots also showed a significant decrease under bottom cooling conditions for both agar concentrations. Lipid peroxidation was always lower in shoots cultured with bottom cooling. All the antioxidant enzymatic activities were lower in bottom cooling treatments compared to controls. These results show that the normalization of the environmental conditions in vitro via bottom cooling can prevent the onset of different simultaneous stress reactions concomitant with hyperhydricity. The present work provides for the first time , direct evidence of a reduced H₂O₂ generation in the tissues cultured in bottom cooling able to reduce oxidative stress.     

Hyperhydricity in shoot cultures of Scrophularia yoshimurae can be effectively reduced by ventilation of culture vessels

An effective procedure for obtaining healthy shoots from nodal segments of Scrophularia yoshimurae is described. Nodal segments cultured on Murashige and Skoog's (MS) basal medium supplemented with 1.0 mg L(-1) benzyladenine (BA) and 0.2 mg L(-1) alpha-naphthaleneacetic acid (NAA) induced multiple shoots in conical flasks that differed in the way they were closed and sealed. Hermitically sealed culture vessels resulted in high hyperhydricity/vitrification. High concentrations of ethylene and CO2 were found to accumulate in these vessels. The hyperhydricity of the shoot cultures could be decreased by progressively ventilating the vessels. Exchange of gases was achieved by removing the Parafilm sealing without affecting sterility. This reduced the hyperhydricity rate and gave a good recovery of vitrified shoots, but resulted in decreased proliferation and a dehydration of proliferating nodal segments and the culture medium. The best number of normal shoots was observed when the parafilm was removed for gaseous exchange after four weeks of culture incubation. The results show that hyperhydricity in shoot cultures of S. yoshimurae could be prevented by sufficient gas exchange during culture.     

A micropropagation protocol forCinnamomum camphora

Summary A micropropagation protocol was developed forCinnamomum 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 N₆-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 EM₂ 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. EM₂ 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 μM BA and 18 per segment when further supplemented with 1000 mg EM₂ per I. Rooting of shoots occurred best when supplemented solely with 0.54 μM NAA, averaging 7 roots per shoot in 4 wk. Ninety percent of rooted shoots survived transfer to the greenhouse.     

Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots

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'.     

Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of <Emphasis Type="Italic">Aloe polyphylla</Emphasis>

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 NH₄ ⁺ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N⁶-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH₄ ⁺, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH₄ ⁺ (10.3 mM). However, in the presence of higher NH₄ ⁺ 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 NH₄ ⁺ 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.     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

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 N⁶-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.     

Analysis of ultrastructure and reactive oxygen species of hyperhydric garlic (Allium sativum L.) shoots

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 O₂ generation rate and H₂O₂ 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.     

Red and Blue Light Emitting Diodes (LEDs) Participate in Mitigation of Hyperhydricity in In Vitro-Grown Carnation Genotypes (<Emphasis Type="Italic">Dianthus Caryophyllus</Emphasis>)

The present study was to determine the factors that can reduce hyperhydricity in in vitro-propagated carnation genotypes. The carnation genotypes (Green Beauty, Purple Beauty, and Inca Magic) were grown in vitro under normal and hyperhydric conditions in white fluorescent light (FL) in which half of the hyperhydric plants were grown in red and blue LEDs (light emitting diodes). It was observed that hyperhydricity leads to oxidative stress in terms of TBARS (thiobarbituric acid reactive substances) content, whereas stress was alleviated by R (red) and B (blue) LEDs. The multiprotein complex proteins such as ATPase (RCI?+?LHC1) PSII-core dimer, PSII-monomer/ATPs synthase, and PSII-monomer/cyt b₆f had decreased levels in hyperhydric conditions grown in white FL; however, the expression level of these photosynthetic proteins was retained in hyperhydric plants grown in R and B LEDs. Moreover, the immunoblots of two photosynthetic proteins (PsaA and PsbA) and stress-responsive proteins such as superoxide dismutase, ascorbate peroxidase, and catalase showed recovery of hyperhydricity in carnation genotypes grown in R and B LEDs. Our present study signifies that red (R) and blue light (B) LEDs reduced the hyperhydricity to control levels by maintaining the composition of thylakoid proteins and antioxidative defense mechanisms in carnation genotypes.     

AgNO<Subscript>3</Subscript> prevents the occurrence of hyperhydricity in <Emphasis Type="Italic">Dianthus chinensis</Emphasis> L. by enhancing water loss and antioxidant capacity

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 (AgNO₃) has been shown to revert hyperhydric plantlets to a normal state. Nevertheless, the effect of AgNO₃ 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 AgNO₃. 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 AgNO₃. Compared with the plantlets cultured in HIM alone, the plantlets cultured in HIM containing AgNO₃ displayed dramatic decreases in water content, ethylene content, and reactive oxygen species (ROS) production (particularly regarding H₂O₂ 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.     

Somatic embryogenesis of Agave victoria-reginae Moore

Plant regeneration through direct somatic embryogenesis of leaf blade explants from in vitro propagated plants of Agave victoria-reginae Moore, is described. Somatic embryogenesis was evident in a 6-week period on agarsolidified MS medium supplemented with L2 vitamins and 2,4-dichlorophenoxyacetic acid (1,4 µM), and germination of somatic embryos was achieved after 8 weeks on half-strength MS medium and 4 weeks on half-strength SH medium, both lacking growth regulators. Hyperhydricity of somatic embryos and plantlets was reduced by the use of vented culture vessel lids during the last 4 weeks on SH medium. Shoot proliferation was obtained, and hyperhydricity was eliminated on a modified MS medium (with NH₄NO₃ reduced to 5 mN) supplemented with kinetin (4.6 µM) and 1-naphthaleneacetic acid (1.6 µM) and the use of vented culture vessel lids.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog - LOG-1 MS modified medium by Castro-Concha et al. (1990) - L2 Phillips and Collins (1979) vitamins - SH Schenk and Hildebrandt     

Effects of culture medium composition and PEG on hyperhydricity in Dendrobium officinale

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.

     

Differences between Dianthus caryophyllus L. cultivar in in vitro growth and morphogenesis are related to their ethylene production

The involvement of ethylene in the vitro development of shoots from nodal segments of two cultivars of carnation (Dianthus caryophyllus L.) was studied. Shoots of cv. Barbaret Antares showed low hyperhydricity in contrast with the high levels showed by cv. Barbaret Tanga when both were cultured in airtight culture vessels. Longer shoots were produced, in both cases, when the rate of gas exchange in the culture vessel was increased by using vented closures, which also prevented hyperhydricity and increased the multiplication coefficient in cultures of Barbaret Tanga.The two cultivars produced ethylene throughout the culture period but, a higher amount was produced during the first, second and fourth weeks in culture by the cultivar more sensitive to ventilation (Barbaret Tanga). Trapping ethylene did not produce any effect on cv. Barbaret Antares but improved the quality of cv. Barbaret Tanga explants, decreasing hyperhydricity and increasing the number of shoots, the length of the main shoot and the multiplication coefficient. These effects were more marked when ethylene was trapped during the first two weeks in culture.     

Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots

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 μm²). 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.     

Restricting hyperhydricity of lettuce using screen raft and water absorbent resin

A method for reducing hyperhydricity of lettuce shoots regenerated from culture on liquid and gelled media is described. In the case of liquid medium, hyperhydricity could be decreased by 30% by using a filter paper raft or a polyester screen raft to prevent immersion of inoculum in the culture medium. In the case of gelled medium, replacing agar with the water-absorbent resin SuperSorb could successfully prevent hyperhydricity without depressing organogenesis.