Response of <Emphasis Type="Italic">Dionaea muscipula</Emphasis> J. Ellis to light stress in in vitro: physiological study

Hyperhydricity can cause significant economic loss for the micro-propagation industry that produces blueberry. In order to predict and control the occurrence of hyperhydricity, better understanding of the anatomical and physiological features of hyperhydric plantlets is required. In this study, we investigated the ultrastructural and physiological changes associated with hyperhydric blueberry plantlets. Compared to normal plantlets, hyperhydric plantlets exhibited reduced cell wall thickness, damaged membrane and guard cell structure, decreased number of mitochondria and starch granule, higher cell vacuolation, more intercellular spaces, and collapse of vascular tissues. In addition, excessive accumulation of reactive oxygen species (ROS) and ethylene, decreased stomatal aperture and water loss, as well as abnormity of stomatal movement were also evident in the hyperhydric plantlets. The results suggested that excessive ethylene and ROS produced in response to the stress arising from in vitro culture could lead to abnormal stomatal closure, causing the accumulation of water in the tissues. This would lead to subsequent induction of oxidative stress (due to hypoxia) and cell damage, especially guard cell structure, eventually giving rise to the symptoms of hyperhydricity. Reducing the content of ethylene and ROS, and protecting the structure and function of the stomata could be considered as potential strategies for inhibiting hyperhydricity or restoring the hyperhydric plants to their normal state.     

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.     

Hyperhydricity in pepper plants regenerated in vitro: involvement of BiP (Binding Protein) and ultrastructural aspects

 Hyperhydricity in regenerated pepper plants was monitored by the induction of the ER-luminal resident protein, as observed by immunoblotting. Immunoblotting of total protein using an anti-soybean BiP serum indicated that the induction and accumulation of an 80-kDa protein was related to BiP (Binding protein), a 78-kDa ER-resident molecular chaperone. The anti-BiP serum cross-reacted with an 80-kDa protein which was significantly induced by hyperhydricity. Based on similar molecular weight and immunological reactivity we concluded that the 80-kDa protein induced in hyperhydric plants is a BiP homologue. The ultrastructural organisation of leaves in non-hyperhydric and hyperhydric pepper (Capsicum annuum L.) plants was investigated with the aim of identifying the subcellular changes associated with this phenomenon. In non-hyperhydric leaves the chloroplasts of the palisade cells had normally developed thylakoids and grana and a low accumulation or absence of starch grains and plastoglobules. In the hyperhydric plants, however, the chloroplasts exhibited thylakoid disorganisation, low grana number, an accumulation of large starch grains and a low accumulation or absence of plastoglobules. Although the structure of mitochondria and peroxisomes did not change in hyperhydric plants, the number of peroxisomes did increase. Received: 23 July 1998 / Revision received: 26 February 1999 / Accepted: 17 March 1999     

The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid-cultured Narcissus

 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     

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.

     

Ancymidol-enhanced hyperhydric malformation in relation to gibberellin and oxidative stress in liquid-cultured <Emphasis Type="Italic">Narcissus</Emphasis> leaves

Summary The growth retardant ancymidol inhibited gibberellin biosynthesis and enhanced hyperhydric malformation of Narcissus leaf sections cultured in liquid medium. Superoxide dismutase activities were examined by spectrophotometry and native polyacrylamide gel analysis, and gibberellin and hydrogen peroxide levels were determined spectrophotometrically in either hyperhydric or non-hyperhydric leaf sections. In ancymidol-treated hyperhydric leaf sections, superoxide dismutase activity and hydrogen peroxide levels were higher during the initial culture period, when hyperhydric malformation occurred, than in control untreated leaf sections. At a later stage, when the meristematic centers started to form on ancymidol-enhanced hyperhydric leaf sections, superoxide dismutase activity, hydrogen peroxide, and gibberellin levels were significantly lower in hyperhydric leaf sections than in non-treated leaf sections. The changes in superoxide dismutase activities, hydrogen peroxide, and gibberellin levels appeared to be related to hyperhydric malformation and meristematic center initiation.     

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.     

Carbohydrate,metabolic, and osmotic changes in scaled-up liquid cultures of <Emphasis Type="Italic">Narcissus</Emphasis> leaves

Summary The use of scaled-up liquid cultures could be an efficient system for mass propagation of Narcissus, as it can greatly reduce the costs involved with manual handling. Induction of hyperhydric meristematic leaf section clusters and proliferation were carried out in an ancymidol (ANC)-containing liquid medium in flasks and disposable presterilized plastic bioreactors. Non-hyperhydric bulblets started to develop from hyperhydric meristematic leaf section clusters after subculture on a 0.8% agar strength medium, and young bulbs formed after 10 mo. in vivo acclimatization with a 98% survival rate. The present study reveals that in Narcissus leaf sections cultured in liquid medium, morphogenetic changes in leaf sections were associated with metabolic changes. The changes in carbohydrate, protein, and water potential of the liquid media and leaf sections were found to be closely related to meristematic center initiation on Narcissus hyperhydric leaf sections. Starch, sucrose, and glucose were significantly higher in the hyperhydric leaf sections cultured in ANC medium. The water potential was signifieantly higher in ANC-treated leaf sections and significantly lower in the medium containing ANC, at the stage shortly before or after hyperhydricity and meristematic centers hegan forming on the leaf sections. A 30kDa protein was found to be present in the hyperhydric leaf sections. Based on the present study, a largescale micropropagation protocol of Narcissus in agar and liquid cultures is proposed.     

Lipid peroxidation and antioxidant enzyme activities of Euphorbia millii hyperhydric shoots

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H₂O₂ from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.     

Sucrose and jasmonic acid interact in photosynthetic pigment metabolism and development of potato (Solanum tuberosum L. cv. Sante) grown in vitro

Potato (Solanum tuberosum L., cv. Sante) plantlets grown from stem node culture on medium supplemented with 90 mM sucrose accumulated lower amounts of photosynthetic pigments per mg dry weight in comparison to those grown on 30 mM sucrose. Addition of 0.1, 1 or 10 µM jasmonic acid (JA) to the medium resulted in a decrease of chlorophylls and carotenoids in the plantlets grown on either sucrose concentration. JA treatment induced de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin only in those plantlets grown on a higher amount of sucrose in which hyperhydric symptoms were observed. The synergistic effect of JA and sucrose was clearly demonstrated in the plantlets grown on 90 mM sucrose and 1 µM JA. This was possibly due to overaccumulation of sucrose, the consequence of the most developed root system, and/or to stimulated water and solute transport by other mechanisms.     

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.     

Genetic transformation through the use of hyperhydric tobacco meristems

Exposed shoot meristems from normal and hyperhydric (vitrified) tobacco, Nicotiana tabacum, were bombarded with gold particles either coated with plasmid DNA containing neomycin phosphotransferase (NPTII), rolC and -glucuronidase (GUS) genes (plasmid pGA-GUSGFrolC) or left uncoated. Meristems bombarded with uncoated particles were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGA-GUSGFrolC. Whole-plant transformants were produced from 4 of 40 hyperhydric meristems bombarded with uncoated particles followed by co-cultivation with A. tumefaciens. One transgenic plant was obtained from 40 normal, non-hyperhydric meristems treated. Transformation was verified by growth on kanamycin-containing medium, GUS assays, PCR, and Southern analysis. The plants tested through Southern analysis appeared to have 2 or more copies of the transgene insert. Seeds obtained from self-pollination of these transgenic plants segregated 3:1 or 15:1 (kanamycin resistant:sensitive) when germinated on medium containing 100 mg/l kanamycin, indicating transfer of foreign genes through the sexual cycle. Whole-plant transformants were not produced from 50 normal tobacco meristems bombarded with plasmid-coated gold particles and not exposed to engineered A. tumefaciens, but 1 plant of 60 bombarded hyperhydric meristems produced transgenic roots, the result of a chimera. We suggest that hyperhydric meristems are more readily transformed.     

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.     

Proteomic and Antioxidant Analysis Elucidates the Underlying Mechanism of Tolerance to Hyperhydricity Stress in In Vitro Shoot Cultures of <Emphasis Type="Italic">Dianthus caryophyllus</Emphasis>

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.     

Micropropagation of <Emphasis Type="Italic">Cotoneaster wilsonii</Emphasis> Nakai—a rare endemic ornamental plant

A simple and efficient micropropagation system was developed for Cotoneaster wilsonii through node and shoot tip explants obtained from mature field-grown plants. Of the two explants, node explants were found to be the most effective for axillary shoot proliferation. The highest frequency of shoot induction was achieved when nodal explants were incubated on Murashige and Skoog (MS) medium supplemented with 0.5 mg L⁻¹ thidiazuron (TDZ) and 0.1 mg L⁻¹ α- naphthaleneacetic acid (NAA) with an average of 34 shoots per explant. The microshoots were separated from the multiple shoots and subcultured on MS medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar for further shoot growth. Maximum rooting was obtained on half-strength MS medium supplemented with 0.5 mg L⁻¹ indole-3-butyric acid (IBA). The in vitro-grown plantlets were successfully acclimatized in a glasshouse with 98% of survival. High concentrations of TDZ (1.5–2.0 mg L⁻¹) and repeated subcultures resulted hyperhydric shoots. Supplementation of the culture medium with silicon significantly reduced the induction of hyperhydric shoots. Increasing silicon concentration significantly decreased malondialdehyde content of the regenerated shoots. Data indicate that addition of silicon to the culture medium can effectively control hyperhydricity.     

Zeatin and TDZ-induced Shoot Proliferation and Use of Bioreactor in Clonal Propagation of Medicinal Herb, Roseroot (Rhodiola rosea L)

A procedure for in vitro propagation of roseroots (Rhodiola rosea L), a medicinal plant, was developed using a RITA bioreactor system containing liquid medium, combined with a gelled medium. Wild roseroot clones: ‘RCi’, ‘RC2’ and ‘RC3’ were established on a basal medium (BM) from in vitro-germinated seedlings on half-strength Murashige and Skoog (MS) salts. TDZ at 2–4 μM supported shoot proliferation but inhibited shoot elongation of ‘RCi’ shoots on gelled medium. Clones differed significantly with respect to multiplication rate with ‘RCi’ producing the most shoots per explant on gelled BM with 2 μM zeatin. In a bioreactor system, TDZ supported rapid shoot proliferation at lower concentration (0.5 μM) but induced hyperhydricity at more than 0.5 μM. Bioreactor-multiplied hyperhydric shoots of all clones when transferred to gelled medium containing 1–2 μM zeatin produced normal shoots within 4 wk of culture. Shoots were rooted in vitro on BM void of growth regulators. Almost all (9U to 95%) in vitro plantlets survived when transferred to potting medium.     

A Study on Comparative Morphology of Normal and Hyperhydric Stems and leaves from Sophora japonica Plantlets Cultured in Vitro

A large numar of plantlets were obtained from cotyledon explants of Sophom japonica L. cultured in vitro. They could be classified into 3 kinds according to their morphological characteristics, viz. the normal plantlets, the hyperhydric planfiets,and the intermediate state between the two or the sub-hyperhydric type. The free water content was more than 79% in the hyperhydric shoots,and 70% in the sub-hyperhydric shoots,while less than 50% in the normal shoots. The surface anatomy of normal, sub-hyperhydric and hyperhydric stems and leaves of the plantlets were compared by scanning electron microscopy. The surface structure of the normal plantlets was similar to those found in field-grown plants,but great change occurred in that of hyperhydric and the sub-hyperhydric plantlets. The stems and leaves surface of the hyperhydric and sub-hyperhydric plantlets appeared to be uneven, wrinkled, brittle and translucent and besides the leaves were thick, curled with a reduced surface area. There was little or no epicuticular wax on the surface of epidermal cells which had irregular shapes and patterns. All leaves were amphistomatic and the stomatal density, size and degree of opening were obviously bigger in the sub-hyperhydric and hyperhydric leaves than in the normal ones. Normal stomata had kidney-shaped guard cells and resembled closely those found in the feild-grown plants, whereas abnormal stomata had deformed guard cells. All of the morphological characteristics mentioned above indicated that the sub-hyperhydric and hyperhydric shoots bended to lose their water easily and resulted in desiccation, which might be one of the major causes of failure to transfer sub-hyperhydric and hyperhydric plantlets to soil.     

Natural ventilation effectively reduces hyperhydricity in shoot cultures of <Emphasis Type="Italic">Aloe polyphylla</Emphasis> Schönland ex Pillans

An investigation into the role of ventilation to reduce hyperhydricity in tissue cultures of Aloe polyphylla Schönland ex Pillans revealed that gaseous exchange between the in-vitro atmosphere and the outside environment is an essential prerequisite for controlling this disorder. In closed culture vessels, hyperhydricity affected as much as 84% of the newly-formed shoots on media gelled with gelrite. The leaves of hyperhydric shoots had a bright green colour, smooth epidermis and large, open stomata. Gaseous exchange was promoted by using modified lids with a hole covered with polyester or cotton mesh. In ventilated cultures, hyperhydricity was completely eliminated irrespective of the type of gelling agent used. Natural ventilation was further advantageous for the microplants in terms of leaf chlorophyll content as well as the deposition of epicuticular wax, indicating the onset of mechanisms that regulate water loss from the explants. Although culture ventilation was negatively correlated to the regeneration rate and shoot growth, it has the potential to control the appearance of abnormal phenotypes and can be easily adopted for routine A. polyphylla propagation in vitro.     

Somatic embryogenesis,plant regeneration,and the evaluation of camptothecin content in <Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

Summary Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.     

Sub-cellular location of H2O2, peroxidases and pectin epitopes in control and hyperhydric shoots of carnation

In carnation shoots (Dianthus caryophyllus cv. Killer), hyperhydricity was induced in in vitro culture using a low agar concentration. Using transmission electron microscopy, cytochemical techniques and immunolocation of JIM5 and JIM7 pectin epitopes, we followed the sub-cellular modifications of cell walls in relation to peroxidase activity and hydrogen peroxide accumulation during hyperhydricity induction. Peroxidase activity revealed a significant induction of the stomatal and epidermal cells as well as of the intercellular spaces of hyperhydric leaves. Similarly, hydrogen peroxide accumulated in the epidermal cell walls and the intercellular spaces of hyperhydric leaves. Immunolocation of an epitope recognised by the JIM5 antibody revealed the main unesterified nature of the cell walls. Such an epitope was located in the epidermal cell walls as well as in the corners of cell junctions in control leaves. However, hyperhydric leaves showed a total reduction of JIM5 labelling in the corners of cell junctions and a significant reduction of the intercellular spaces and the middle lamella. Highly-methylsterified pectin, recognised by the JIM7 antibody, was present to a slight extent in cell walls in control and hyperhydric leaves. We propose that the altered anatomy observed in hyperhydric carnation leaves could be regulated by the concomitant actions of pectin methyl esterases and free radicals, modifying the structure of the pectin and polysaccharides of the cell walls.