Special symposium: In vitro plant recalcitrance do free radicals have a role in plant tissue culture recalcitrance?

Summary Free radicals have an important role in the metabolism and development of aerobic organisms; however, their uncontrolled production leads to oxidative stress. This paper explores the possibility that free radical mediated stress has a role in tissue culture recalcitrance. In the context of this paper, recalcitrance is considered to be the inabilit of plant tissue cultures to respond to culture manipulations; in its broadest terms, this study also concerns the time-related decline (i.e. in vitro aging) and loss of morphogenetic competence and totipotent capacity. Studies on a diverse range of in vitro plant systems have shown that tissue cultures produce free radicals, lipid peroxides and toxic, aldehydic lipid peroxidation products. Levels of these compounds vary in response to different tissue culture manipulations, but their production is enhanced during dedifferentation and antioxidant profiles also vary throughout different phases of culture. A hypothesis is presented which suggests that tissue culture manipulations cause major metabolic and developmental changes, some of which may predispose in vitro cultures to increased free radical formation. If antioxidant protection is compromised, oxidative stress ensues and free radicals and their reaction products react with macromolecules such as DNA, proteins and enzymes, causing cellular dysfuction and as a result, the cultures become recalcitrant.     

Alternative statistical analyses for micropropagation: A practical case of proliferation and rooting phases in <Emphasis Type="Italic">Viburnum opulus</Emphasis>

Summary The kinds of data obtained in micropropagation studies are very often problematic, since they do not follow continous distribution and observations through culture vessels complicate measurement. Accordingly, standard analyses are often used, leading to misinterpretation of results. In this paper, we present a study of Viburnum opulus micropropagation using planned contrasts and fitting regression models in generalized linear models as an alternative statistical analysis of micropropagation results, and compare the results with that of traditional ANOVA. The advantages and possibilities of the alternative data analyses in plant tissue culture are discussed.     

Agronomic evaluation of tissue-culture-derived soybean plants

Summary Genetic alterations of regenerated plants based on the tissue culture process (somaclonal variation) have become common for many plant species including soybean [Glycine max (L.) Merr.]. The objective of this study was to test for the presence of tissue-culture-derived genetic variation in eight agronomic traits in homozygous progeny regenerated by organogenesis using the commercially important cultivar Asgrow A3127. A total of 86 lines derived by repeated self-pollination of nine regenerated plants was grown in two locations for 2 years. When compared to the unregenerated parent, statistically significant variation (P<0.05) was found for maturity, lodging, height, seed protein and oil, but not for seed quality, seed weight, or seed yield. All of the variation noted was beneficial and did not involve decreased yield. Since the differences were not large, the results indicate that the tissue culture process is not necessarily detrimental to plant performance, which is an important consideration since tissue culture techniques are used in many genetic engineering methods.     

Digital photography for rhinoplasty

Standardized, high-quality, preoperative photographs of the nose are critical for preoperative rhinoplasty planning, comparative postoperative assessment, and demonstration of surgical results. To produce these high-quality, reproducible photographs, it is essential to standardize lighting, to properly position the patient in standard views, to avoid lens distortion, and to maintain consistent camera-to-subject distances. Traditional photographic standards have been well documented in the literature; however, most do not address digital photography, and none address digital photography for rhinoplasty. Certain variables in digital photography that are not present in 35-mm photography can be critical to the appearance of the final image. Variables such as image color and contrast (which usually vary between digital cameras), focal length differences between 35-mm and most digital cameras, the effect of resolution and compression on image quality, and the effect of the printing method used can affect the appearance of the external anatomy of the nose in the final print or image. Lack of detail in the external nasal anatomy becomes an issue if the surgeon uses the photograph intraoperatively for reference, as the authors do. Initially, the authors experienced difficulties with observing subtleties in the tip-defining points and tip anatomy using digital photography when compared with our traditional methods of 35-mm photography. The lack of detail in the external anatomy was most prevalent in the frontal and basal views. Thus, the authors have since tailored their photographic methods to document the rhinoplasty patient to maximize the visual information of the external nasal anatomy in the photographic and the printed image. This article is intended to review the photographic principles for standardized rhinoplasty photography, address the additional considerations necessary when using digital photography, discuss the printing variables that can affect overall quality of the printed image, and discuss the authors' new method of photographing the rhinoplasty patient.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of some important chinese medicinal plants and their sustainable usage

Summary Medicinal plants are valuable sources of medicinal and many other pharmaceutical products. The conventional propagation method is the principal means of propagation and takes a long time for multiplication because of a low rate of fruit set, and/or poor germination and also sometimes clonal uniformity is not maintained through seeds. The plants used in the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increase in the demand for the erude drugs, the plants are being overexploited, threatening the survival of many rate species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. The purpose of the present review is to focus the application of tissue culture technology for in vitro propagation via somatic embryogenesis and organogenesis and the cell suspension culture with suitable examples reported earlier. An overview of tissue culture studies on important Chinese medicinal plants and related species is presented.     

The histogenesis of somaclones from tomato (Lycopersicon esculentum Mill.) cotyledons

Summary A histological study ofin vitro cultured cotyledonary expiants of tomato (Lycopersicon esculentum) was performed in order to determine the site (differentiated tissue or developing callus) and the mode of plant regeneration.Results have shown that callus develops at the excision sites of cotyledonary expiants and that shoots are formed exclusively within the unorganized callus: excision areas are the only morphogenetic sites and the proximal excision is the preferred site for plant regeneration.Shoots differentiate by organogenesis within the superficial region of the callus. Few neocambial cells cooperate in the neoformation. Origin from a single cell is highly unlikely since rarely observed single activated cells never developed into shoots.Regenerated plants may be chimeras if invitro culture induces genetic diversity in the initial cells.Abbreviations IAA Indole-3-acetic acid - c callus - d vegetative dome - s shoot - ad adaxial - ab abaxial - t tracheid - p parenchyma - S sieve tube     

Effect of Chlorsulfuron on Morphogenetic and Disordered Cell Division in Cultures of Passiflora edulis

We examined the effects of a sulfonylurea herbicide, chlorsulfuron, which is known as a potent inhibitor of plant cell division, on morphogenetic cell division and disorganized cell division using the culture system of multiple shoot primordia and callus of Passiflora edulis. The multiple shoot primordia tissue treated with chlorsulfuron failed to achieve shoot morphogenesis, and a large part of the tissue was necrotized during the posttreatment culture, even when it was washed and transferred to chlorsulfuron-free medium. The inhibition of Passiflora shoot morphogenesis by chlorsulfuron was not reversed by the simultaneous addition of branched amino acids, which are known to reverse the inhibitory effect of chlorsulfuron. In contrast, the same treatment of chlorsulfuron on the callus did not kill the cells, although the growth resumption was retarded by a prolonged lag period. The addition of branched amino acids enhanced the recovery growth of the chlorsulfuron-treated callus. These results suggest that the inhibition of disorganized cell division (callus growth) by chlorsulfuron is reversible, whereas morphogenetic cell division (shoot morphogenesis), which is under complex regulation, is inhibited irreversibly by chlorsulfuron. Qualitative differences between morphogenetic cell division and disordered simple proliferative cell division are discussed. Received November 17, 1997; accepted June 4, 1998     

Micropropagation of Ranunculus Asiaticus: A Review and Perspectives

Ranunculus asiaticus is an important ornamental species mainly cultivated in the countries surrounding the Mediterranean sea. So far the multiplication of this plant has been mainly carried out by seed and rhizome division; however, these systems present many drawbacks. Tissue culture is an attractive alternative for accelerated propagation of selected and indexed genotypes. In this paper we review the work carried out on in vitro culture of R. asiaticus and present a flow chart for its commercial production, using axillary budding and embryogenesis. Although the price of micropropagated plants is higher compared to traditional material (seedlings and rhizomes from seed populations), it should be considered that tissue cultured plants have a better rhizome yield per plant; moreover, the tissue culture approach allows to offer clonal material of selected lines.     

Induced responses in Ipomoea hederacea: simulated mammalian herbivory induces resistance and susceptibility to insect herbivores

Multispecies interactions between plants and natural enemies are ubiquitous, and often lead to diffuse interactions between plants and their herbivores. Non-specific induced responses, where responses induced by one species affect other species, are one potential mechanism generating diffuse interactions. Using 57 inbred lines of the Ivyleaf morning glory, Ipomoea hederacea, in a greenhouse experiment, we examined whether simulated mammalian herbivory induced responses that could affect plant resistance to the generalist insect herbivore, Spodoptera exigua. Inbred lines were highly variable for induced responses, ranging from induced resistance to induced susceptibility, with the rank-order for resistance in inbred lines changing between clipping and control treatments. We failed to detect significant genetic correlations between induced responses and trichome density, or that clipping modified the negative relationship between trichome density and Spodoptera exigua consumption and biomass. Our results suggest that non-specific induced responses can mediate the diffuse evolutionary relationship between I. hederacea and its herbivores, and that genetic variation in induced responses are an important component of this interaction. Handling Error: Heikki Hokkanen     

Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5 non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions –623 to –471 and from –471 to –341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position –1185 to –341, although at different levels, maintained the same pattern of GUS expression — maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from –341 to –306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.     

Plant tissue culture. Biotechnology. Milestones

Summary The progress in the development of the technologies of plant tissue and cell culture over the past four decades has been remarkable. This article covers my personal reflections on the various topics and is based on my involvement in the field during that period. There are three fundamental technologies which constitute most of what is referred to as plant in vitro technologies or tissue culture. The origin and some of the key persons involved in the development of each of these procedures will be discussed. The technology that is most common is growing plant tissue on gel-solidified nutrient media. That technology is being used in the most vital procedures, namely the regeneration of plants from cultured cells. The culture of plant cells in liquid suspension was developed very shortly after that, and has become a very effective technology for plant regeneration by somatic embryogenesis. The method of meristem culture arose out of a need for developing plants that were virus-free. In many species the technique is now being used to produce virus-free crop plants. Another important technology is the culture of anthers and microspores for producing haploid and homozygous plants. Included with plant tissue culture is the development of the plant protoplast and cell fusion technologies for the production of new plant hybrids. The final aspect of the development concerns the integration of tissue culture with molecular genetics, which has developed into the rapidly expanding field of biotechnology.     

Moderation of morphogenetic and oxidative stress responses in flax in vitro cultures by hydroxynonenal and desferrioxamine

Hypocotyl segments of 7-day old seedlings of flax (Linum usitatissimum L.) cultivars Atalante, Flanders, Jitka, Szegedi 30 and Super were screened for organogenesis (shoot and root induction) and embryo-like structure production. A non-destructive assay for hydroxyl radicals (*OH), utilising DMSO as a radical trap, was used to determine *OH formation during tissue culture and morphogenesis. Desferrioxamine, an inhibitor of Fenton reaction, and 4-hydroxy-2-nonenal, a cytotoxic Lipid peroxidation product, were exogenously applied to flax cultures to determine the effect of antioxidative and prooxidative status on morphogenetic responses induced through the exogenous application of plant growth regulators. Flax genotypes varied in their response to treatments after exposure to different plant hormones. Hydroxyl radical (*OH) formation correlated with morphogenetic responses and this was affected by plant hormones. Desferrioxamine and 4-hydroxy-2-nonenal also moderated morphogenetic responses and influenced hydroxyl radical formation during in vitro propagation.     

Integrated Application of Physiological and Molecular Methods to Forecast Determinative Morphogenetic Events in Tissue Cultured Tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. cv Samsun) Leaf Discs

The purpose of this study was to search for physiological parameters that provide an early indication of the morphogenetic response of leaf disc explants to different tissue culture-level manipulations in order to design an accelerated optimisation process for this technology. Tobacco (Nicotiana tabacum L. cv. Samsun) was chosen as model in our studies, because this is still one of the most widely used species in experimental plant biology and detailed knowledge is available from its tissue culture system. Large numbers of physiological markers at the whole plant level (rates of photosynthesis and respiration) and cellular level ‘fitness’ (degree of DNA methylation) were measured together with the concentrations of the most abundant metabolites in photosynthetic carbohydrate metabolism, ATP and protein synthesis in leaf discs induced either for callus development or for shoot differentiation. As a result of the above examinations, we are able to show that the efficiency of photosynthesis, the rate of respiration and the degree of DNA methylation can be used as early markers for the changes that precede the appearance of in vitro morphogenesis. By examining these parameters, we were able to predict early ontogenesis (within 48 h) and the optimal concentrations of growth regulators needed to achieve either shoot differentiation or callus development. Collaborator via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems     

A comparison of tissue culture response between related tetraploid and dihaploid S. tuberosum genotypes

The response to tissue culture of a series of related, agronomically useful, dihaploid (2n=2x=24) and tetraploid (2n=4x=48) S. tuberosum genotypes was assessed by regenerating shoots from leaf explants. Dihaploid genotypes that showed superior responses to their tetraploid parents were identified. Large differences in tissue culture response were also found between dihaploid genotypes derived from the same tetraploid parents. These results indicate that it should be possible to select agronomically useful dihaploid genotypes with good tissue culture responses for use in genetic manipulation experiments. Possible factors determining tissue culture response in S. tuberosum are discussed.     

Neo-formation of flower buds and other morphogenetic responses in tissue cultures of Melia azedarach

Melia azedarach has great interest because of its insecticidal properties. Recently, the occurrence of precocious flowering in tissue cultures of this species was reported. This paper describes some in vitro morphogenetic responses using hypocotyl segments as explants and MS basal medium. Amongst the results we report are: (a) in basal medium, 5% of the explants neo-formed floral buds and flowers, and 80% formed vegetative shoots; (b) flower neo-formation could not be controlled or increased by addition of benzyladenine, or lowering the nitrogen level; (c) benzyladenine increased the regeneration of vegetative shoots; (d) compact green calluses were eventually formed in basal medium, and vigorous friable calluses can be easily induced with 0.5 mM 2,4-D; (e) green calluses could be subcultured and regenerated into plants, and, from friable calluses, cell suspensions were started; (f) histological studies showed that neo-formations originate in the wound tissue or from the inner tissue of the hypocotyl.     

紫茉莉不定芽诱导和植株再生

紫茉莉(Mirabilis jalapa)观赏价值较高,是一种重要的污染修复植物.组织培养技术为植物品种改良和选育的重要途径,但紫茉莉离体快繁方面的研究尚未见有相关报道.该研究以紫茉莉叶片和茎段为外植体,通过观察和统计外植体愈伤组织和不定芽的诱导情况,分析不同植物生长物质对紫茉莉植株再生的影响.结果表明:紫茉莉带芽茎段比较适合丛生芽的诱导,当带芽茎段在MS+1.0 mg·L-16-BA+1.5 mg·L-1 KT+1.0 mg·L-1 NAA+0.05 mg·L-1 TDZ培养基中培养时,不定芽的增殖系数较高.无论是MS或1/2MS培养基,都可诱导不定根的产生,其中生根效果较好的培养基为1/2 MS+0.5 mg·L-1 NAA.该研究结果探索了紫茉莉组织培养的最适条件,根据愈伤组织诱导率和不定芽的增殖系数筛选出了适宜不定芽诱导的培养基类型,根据不定芽生根情况确定了最佳的生根诱导培养基,为建立紫茉莉高效稳定的再生和遗传转化体系奠定了基础.     

In vitro reactions of sugarcane (Saccharum SP.) plantlets inoculated with 2 strains of Xanthomonas albilineans (Ashby) Dowson

The symptoms of the leaf scald disease can be reproduced in vitro through the inoculation of sugarcane tissue culture plantlets. The pathogen is detected in the inoculated plantlet and is maintained at the surface of the base of the plantlets grown in vitro. Two strains of X. albilineans belonging to different serovars and lysovars reacted like pathotypes. The importance of the plant incubation temperature is clearly demonstrated. Further, in vitro the disease goes through the same phase of latency as in the field.     

Analogues of auxin modifying growth and development of some monocot and dicot plants

The dependence of morphogenetic processes in the formation of vegetative and generative organs in spring oilseed rape and barley on exogenously applied physiological analogues of auxin: 2,4-D (2,4-dichlorphenoxyacetic acid), NAA (naphthalene-1-acetic acid), TA-12 (1-[2-chloroethoxycarbonyl-methyl]-4-naphthalenesulfonic acid calcium salt) and TA-14 (1-[2-dimethylaminoethoxicarbonylmethyl]naphtalene chlormethylate) were investigated. The experiments were performed with hypocotyl tissue cultures of oilseed rape and barley microspores in vitro. The auxin analogues applied revealed differences of morphogenetic competence in dedifferentiation-redifferentiation processes that occurred in oilseed rape cultures. TA-12 and TA-14 applied together with NAA and BA (6-benzylaminopurine) caused more intensive callus growth in comparison with 2,4-D. Rhizogenesis was induced when 2,4-D was substituted by TA-12. Compound TA-14, unlike TA-12, facilitated the appearance and development of cotyledons in callus tissues. Hower the compounds TA-12 and TA-14 have no positive effect in monocot plant — barly anther culture for callogenesis and regeneration in comparison to indole-3-acetic acid (IAA). TA-14 and TA-12 showed similar but not identical auxin properties and demonstrated high efficiency as modifiers of rape-dicot plant growth and morphogenesis.     

Spatial integration of the partners and heteromorphism of the cyanobacteriumNostoc muscorum CALU 304 in a mixed culture with theRauwolfia tissue

A comparative morphological study was conducted ofNostoc muscorum CALU 304 grown either as a pure culture on standard media or as a mixed culture withRauwolfia callus tissue on a medium for plant tissue cultivation. The interaction of the cyanobacterial and plant partners results in their spatial integration into aggregates of specific anatomy, which arise periodically during the mixed culture growth. The morphology of the cyanobacterial cells varies depending on their localization in the mixed aggregate. The degree of cyanobacterial heteromorphism increases with the time of growth of the association. Evidence of the plant origin of the factors inducing heteromorphic changes inN. muscorum was obtained, as well as evidence indicating that these factors can rapidly diffuse in agarized medium. A conclusion is inferred that the heteromorphic cells correspond to bacterial forms that appear during unbalanced growth as an adaptation to altered environmental conditions.     

Cotton ovule culture: A tool for basic biology, biotechnology and cotton improvement

Summary Nearly 30 years ago the conditions for culturing immature cotton ovules were established to serve as a working research tool for investigating the physiology and biochemistry of fiber development. Not only has this tissue culture method been employed to characterize the biochemistry of plant cell expansion and secondary cell wall synthesis, but ovule cultures have contributed to numerous other aspects of plant cell physiology and development as well. In addition to basic studies on fiber development, cotton ovule cultures have been used to examine plant-fungal interactions, to model low temperature stress responses, to elucidate the pathways responsible for pigment formation in naturally pigmented fiber and to probe how cytoskeletal elements regulate cell wall organization. Success in rescuing Gossypium interspecific hybrids was dependent on ovule culture media formulations that could support early embryo development in ovulo. As tissues produced in culture are analyzed by increasingly more sophisticated techniques, there appear to be some differences between ovule growth in planta and ovule growth in vitro. Discerning how ovule culture fiber development is different from fiber development in field-grown plants can contribute valuable information for crop improvement. Cotton ovule cultures are an especially attractive model system for studying the effects of gravity on cell elongation, cellulose biosynthesis and embryo development and are excellent targets for examining transient expression of introduced gene constructs. With only minor modification, the procedure originally described by C. A. Beasley and I. P. Ting for growing cotton ovules in vitro will continue to be useful research tool for the foreseeable future.