Structural differences between hyperhydric and normal in vitro shoots of <Emphasis Type="Italic">Handroanthus impetiginosus</Emphasis> (Mart. ex DC) Mattos (Bignoniaceae)

The anatomy of normal and hyperhydric shoots (leaves and stems) of in vitro Handroanthus impetiginosus was compared using light microscopy, scanning electron microscopy, and transmission electron microscopy. In contrast to normal shoots, hyperhydric shoots presented numerous anatomical abnormalities at the proliferation stage. Disorganized cortex, epidermal holes, epidermal discontinuity, collapsed cells, and other structural characteristics were observed in hyperhydric shoots. So, by using anatomical analysis of in vitro H. impetiginosus shoots at the proliferation stage, we can predict which plants will survive the rhizogenesis and acclimatization stages.     

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.     

Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress

The physiology of hyperhydricity in relation to oxidative stress, mineral nutrients, antioxidant enzymes and ethylene has been studied in three micropropagated carnation cultivars under experimentally induced hyperhydricity. A marked increase in Fe content in comparison with normal tissues was observed in the hyperhydric tissues from the three cultivars. The levels of ethylene, solute leakage and malondialdehyde content were also significantly higher in the hyperhydric tissues. In relation to the time course of H₂O₂ production measured by fluorescence quenching, a similar trend could be observed for the three cultivars, with a clear increase in the generation of hydrogen peroxide in hyperhydric tissues. The activities of all the antioxidative enzymes studied, except lipoxygenase, were higher in the hyperhydric shoots. Phenylalanine ammonia-lyase (PAL) showed a significant decrease in activity in the hyperhydric tissues in comparison with the controls for the three cultivars. Soluble guaiacol peroxidase had a strong increase in activity in hyperhydric shoots of the three cultivars. These results provide, for the first time, direct evidence of H₂O₂ generation in hyperhydric tissues, characterize the response of the antioxidant system to an oxidative stress during hyperhydricity in carnation leaves and point to the accumulation of toxic forms of oxygen as the inducer of some of the abnormalities observed.     

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.     

槐树试管正常苗与超度含水态苗茎叶的比较形态学研究

槐树(Sophora japonica L.)子叶经培养获得了大量的试管苗,依其形态正常与否可将其分为正常苗、超度含水态苗和介于二者之间的过度含水态苗。其自由水含量明显不同,正常苗低于50％,超度含水态苗高于79％,而过度含水态苗约为70％。以扫描电镜对其茎、叶的形态学结构进行了比较研究,结果表明:正常苗茎、叶表皮结构基本类似于实生苗,而过度及超度含水态苗的茎、叶表皮层结构变异较大。主要表现在其表面凹凸不平,表皮层外稀有或无蜡质存在,表皮细胞形状及排列不规则,叶片近、远轴两面气孔器密度、大小及开度均较正常苗显著增大;保卫细胞形态、结构异常。上述特征,均显示出槐树试管过度及超度含水态苗易失水干化,这可能是其在移栽过程中难以成活的主要原因之一。     

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     

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.     

Hyperhydricity of Prunus avium shoots cultured on gelrite: a controlled stress response.

Hyperhydricity is a physiological disorder frequently affecting shoots vegetatively propagated in vitro. Hyperhydric shoots are characterised by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and a high water content. Hyperhydricity of Prunus avium shoots was expressed in vitro in one multiplication cycle by replacing the gelling agent agar (normal shoots: NS) by gelrite (hyperhydric shoots: HS). P. avium shoots evolving towards the hyperhydric state produced higher amounts of ethylene, polyamines (PAs) and proline, which are substances considered as stress markers. A higher activity of glutathione peroxidase (GPX; EC 1.11.1.9), involved in organic hydroperoxide elimination, suggested an increased production of these compounds in HS. The unchanged free fatty acid composition indicated no HS membrane damages compared to NS. The ploidy level of HS nuclei was not affected, but the bigger size and the lower percentage of nuclei during the S phase suggested a slowing down of the cell cycle. The results argued for a stress response of the HS, but no signs of oxidative damages of lipid membrane and nucleus were observed. The discussion points out paradoxical results in a classical analysis of stress and suggests an alternative way of defense mechanisms in HS, involving homeostatic regulation and controlled degradation processes to maintain integrity and vital functions of the cell.     

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.     

Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff. (Cactaceae) spontaneously produced callus. The habituated callus regenerated normal and hyperhydric shoots without the addition of grown regulators. Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR). Both tumour lines grew vigorously, never expressing any morphogenic potential. In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns. Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants. Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher. Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates. The highest esterase activity was determined in tumours with both substrates. All tissues, except the TR tumour, had higher esterase activity for 2-compared to 1-naphtylacetate. Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.     

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.     

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.     

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.     

A two-step procedure for in vitro multiplication of cloudberry (<Emphasis Type="Italic">Rubus chamaemorus</Emphasis> L.) shoots using bioreactor

Cultures of three cloudberry (Rubus chamaemorus L.) clones collected from natural stands in Newfoundland and Labrador, Canada were established in vitro on a modified cranberry (Vaccinium macrocarpon Ait.) tissue culture medium containing 8.9 μM 6-benzylaminopurine (BAP). Clones were compared for in vitro shoot proliferation on gelled medium supplemented with varying levels of BAP and thidiazuron (TDZ). Addition of 5.8 μM gibberellic acid (GA₃) in 8.9 μM BAP-contained medium improved shoot proliferation. TDZ supported rapid shoot proliferation at low concentration (1.1 μM) but induced 20–30% hyperhydricity in a plastic airlift bioreactor system containing liquid medium. Bioreactor-multiplied hyperhydric shoots were transferred to gelled medium containing 8.9 μM BAP and 5.8 μM GA₃ and produced normal shoots within 4 weeks of culture. Genotypes differed significantly with respect to multiplication rate with ‘C1’ producing the most shoots per explant. Proliferated shoots were rooted on a potting medium with 65–75% of survivability of rooted plants. Present results suggested the possibility of large-scale multiplication of cloudberry shoots in bioreactors.     

Morphology and ultrastructure of Mammillaria gracillis (Cactaceae) in in vitro culture

Morphogenetic status of cactus Mammillaria gracillis Pfeiff. tissue culture was studied by light and electron microscopy. In vitro propagated shoots spontaneously developed callus. This callus regenerated normal and hyperhydric shoots without exogenous hormones. Tumour tissue induced by wild or rooty strains of Agrobacterium tumefaciens never expressed any morphogenetic potential. Light microscopy showed cellular characteristics of morphologically different tissues. Ultrastructural studies revealed changes in plastids: secondary dedifferentiation of mature chloroplasts, thylakoid swelling and disruption, phytoferritin accumulation, plastid elongation and increase in size. Changes in chlorophyll and carotenoid content were in accordance with degradation of the thylakoid system. Plastids were confirmed as very sensitive organelles to an artificial hyperhydric environment as well as to Agrobacteria-mediated cell transformation.     

Growth Conditions in In Vitro Culture Can Induce Oxidative Stress in <Emphasis Type="Italic">Mammillaria gracilis</Emphasis> Tissues

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 (H₂O₂) 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.     

Cytokinins,auxins and activated charcoal affect organogenesis and anatomical characteristics of shoot-tip cultures of lisianthus [<Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> (RAF.) Shinn]

Summary Variants from seed-propagated Lisianthus [Eustoma grandiflorum (Raf.) Shinn] were shoot-tip cultured to observe the effects of cytokinins, auxins and activated charcoal on organogenesis and anatomical characteristics. N⁶-Benzyladenine (BA) and kinetin at high concentrations (13.32–22.2 and 13.94–23.23 μM) resulted in good shoot formation but high percentages of hyperhydric shoots. Increased indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) concentrations favored root formation, while increased naphthaleneacetic acid concentration adversely affected root formation. Both shoot and root development were suppressed by activated charcoal. The highest percentage of regeneration and the largest number of glaucous shoots with an average of 15 shoots per explant after 4 wk of culture were obtained when the shoot tips were cultured on MS (Murashige and Skoog, 1962) medium supplemented with 4.44 μM BA and 1.47–4.92 μMIAA and IBA. In vitro-grown leaves had a higher number of stomata than field-grown leaves but the length and diameter of stomata showed no significant difference between the two types. Field-grown leaves had well-developed epicuticular wax layers. which were not observed on hyperhydric leaves. Hyperthydric plantlets could not survive when transplanted to soil, whereas glaucous plantlets survived in more than 80% of cases. Variation in soil type resulted in a slight difference in plantlet survival. Based on the results of our experiment, this protocol should be useful for the rapid micropropagation of lisianthus.     

A procedure to prevent hyperhydricity in cauliflower axillary shoots

Cauliflower axillary shoots exhibited hyperhydric status when cultured in vitro under usual conditions with growth regulators, resulting in poor survival during transfer to the glass-house. A pretreatment soaking of curd explants in sucrose −2 MPa for 24 h was sufficient to induce axillary bud break. Afterwards, leafy shoots developed on MS medium without any growth regulator. Shoots produced were unhyperhydrated and were successfully transfered for rooting to the glass-house. This simple procedure can be useful in cauliflower breeding programmes where acclimatization is generally a critical stage. This revised version was published online in June 2006 with corrections to the Cover Date.     

麝香石竹玻璃苗与正常苗的生理特性差异

诱导麝香石竹茎段外植体产生不定芽的分化,所得到的正常苗与玻璃苗的生理特性明显有差异。表现在玻璃苗的鲜重、干重、粗纤维和叶绿素含量与正常苗相比显著降低;玻璃苗的可溶性糖含量增加38％,而蔗糖含量下降63%,束缚水含量显著增高,自由水含量明显降低;玻璃苗的淀粉酶总活性也明显升高,碱性和中性区过氧化物酶同工酶活性显著提高而酸性区的同工酶活性有所下降。然而,玻璃苗和正常苗形成时的芽分化频率以及伸长生长量之间无明显区别。结果表明,麝香石竹试管苗的玻璃化可能是在碳水化合物代谢、氮代谢和水分存在状况等发生生理异常的情况下在芽分化启动后的生长过程中发生的,而不是在芽分化启动时已经决定的。