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.     

Heritable variability induced by the in vitro culture and genetic improvement of cultivated plants

Abstract

Genetic variability is found among plants derived from in vitro cultures of somatic cells. A number of different factors, such as the pre-existing genetic variation developed in vivo during tissue differentiation, the variation induced during the in vitro culture and also the selection for specific genotypes during plant regeneration, are considered as possible causes of the phenomenon.

The nature of the genetic changes induced in somaclones (variation in chromosome number, gross and cryptic chromosomal rearrangements, transposition of genetic elements, gene amplification and somatic gene rearrangements) is also discussed.     

Isolation of oxidative stress response mutants in Escherichia coli for their use as a genetic screen to identify new anti-mycobacterials as functional analogues of isoniazid

Summary Tuberculosis is a leading killer disease of the world with increasing mortality due to HIV-infected individuals becoming highly prone to this infection. An attempt has been made in the present work to identify novel plant-derived compounds active against Mycobacterium tuberculosis (MTB) through construction of a target based bio-screen to facilitate rapid screening of anti-TB plant compounds. To achieve this, construction of a genetically modified model system was attempted in fast growing, non-pathogenic, Escherichia coli in which experimental testing is relatively easier and rapid as compared to M. tuberculosis, which is pathogenic and slow growing in nature. The exquisitely high sensitivity of M. tuberculosis to isoniazid (INH) has been attributed to lesions in oxyR, a gene that positively regulates the expression of a set of hydrogen peroxide-inducible genes in E. coli and S. typhimurium. Moreover in the mechanism of emergence of INH resistance in M. tuberculosis, oxidative stress response has been implicated. In this study, mutants of E. coli defective in oxidative stress response function were derived and used to screen plant compounds, which might interfere with the oxidative stress response in MTB. Since MTB is inherently known to be oxyR defective and thus being highly sensitive to INH, mutants defective in oxidative stress response were isolated to construct a model system in E. coli, which is otherwise INH resistant, having functional oxyR. These mutants showed simultaneous sensitivity to oxidative stress-causing agents like hydrogen peroxide and cumene hydroperoxide. To further define the mutational lesions, complementation studies were carried out through mobilization of cloned wild type genes involved in the oxidative stress response and in this way a biological screen was constructed to identify plant compounds/essential oils/extracts/oil components which induce oxidative stress. The positives were finally tested for activity against M. tuberculosis strain H37Rv using the radiometric BACTEC 460 TB system. Interestingly, the bioactives were found to be active against the pathogen with marked potency, as the reduction in δGI values for the identified bioactives against M. tuberculosis were significant. The study demonstrates application of a specific target-based genetic model system in E. coli as a rapid high throughput screen in identifying anti-mycobacterials from plants.     

Assessment of genetic stability in tissue-cultured products and seedlings of Saussurea involucrata by RAPD and ISSR markers

To control the genetic quality during the whole process of tissue culture of the traditional Chinese medicinal plant, Saussurea involucrate Kar. et Kir., DNA polymorphisms and genetic variations were investigated using randomly amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers. The genetic stability/variation in tissue-cultured products, including three calli, three adventitious shoots, regenerated plantlets and 2 year-old regenerated plantlets cultivated in the planting base in Tianshan Mountain, were assessed compared with 1 year-old and 2 year-old seedlings cultivated in the same planting base using aseptic seedlings as reference. Apparent genetic variation was detected in the 11 type of plant materials. The percentages of polymorphic bands in the RAPD and ISSR analysis were, respectively, 35% and 33%. Cluster analysis indicated that the genetic similarity values calculated on the basis of RAPD and ISSR data among the 11 type of plant materials were respectively ranged from 0.823 to 0.995 with a mean of 0.878 and 0.825 to 0.974 with a mean of 0.885, which classified the samples into three groups. The similarity coefficient also revealed that differences among three calli were not remarkable by both RAPD and ISSR analysis, and only chemical components and growth properties needed consideration in the screening of callus used for the next redifferentiation studies. But there are remarkable differences among three adventitious shoots analyzed by ISSR markers. Therefore, RAPD and ISSR markers are efficient tools in genetic variation assessment and quality control in plant tissue culture process.     

A novel ABA-hypersensitive mutant in Arabidopsis defines a genetic locus that confers tolerance to xerothermic stress

Hot and dry air (harmattan or xerothermic climate) greatly inhibits plant growth, particularly flowering and seed setting of crops. Little is known about the mechanism of plant response to this extreme environmental stress due to the lack of valuable genetic resource. Here, we report the isolation and characteristics of a unique Arabidopsis mutant, hat1 (h armattan t olerant 1), which shows high tolerance to hot and dry air. Under normal growth conditions, the mutant does not differ in morphology and soil drought tolerance compared to the wild type. When subjected to high temperature (42°C) and low humidity (10–15%), however, it could survive up to 6 days, while the wild type (Col-0) died after 24 h. The hat1 mutant also exhibits enhanced tolerance to soil drought, but only under xerothermic conditions. Mutant plants tightly close their stomata to retain water under xerothermic stress, and are more tolerant to high salinity at all developmental stages, accumulating less Na⁺ and more K⁺ than wild-type plants during NaCl treatment. Interestingly, hat1 plants are also ABA-hypersensitive. Genetic analysis revealed that the hat1 phenotype is caused by a dominant mutation at a single nuclear locus. Mapping studies indicate that Hat1 is located at an interval of 168 kb on chromosome 5 in which 21 genes are known to be regulated by diverse abiotic stresses. A mutant of this kind, to our knowledge, has not been previously reported. Thus, this report serves as a starting point in the genetic dissection of the plant response to xerothermic stress, and provides physiological and genetic evidence of the existence of a novel abiotic stress response pathway that is also ABA-dependent.     

Plant cell and biotechnology studies in <Emphasis Type="Italic">Linum usitatissimum</Emphasis> – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.     

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> during β-carotene production in submerged fermentation

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during β-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of β-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during β-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased β-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.     

Genetic analysis of anther and leaf disc culture in two clones of Solanum chacoense Bitt. and their reciprocal hybrids

Two diploid clones of self-incompatible Solanum chacoense Bitt. with androgenetic ability were tested for anther and leaf disc culture response together with eight of their reciprocal F1 hybrids. Large differences were found among genotypes in frequency of anther induction as well as in the phase of plant regeneration. Anthers harvested in June showed a significantly higher percentage of response (17.5%) at the induction phase than those collected in July (13.8%) or August (12.7%). The lowest induction frequency was observed in May (7.3%). By contrast, plant regeneration from induced anthers did not vary during this time. Genotypic differences were also observed in leaf disc response. The two parental clones and two of their hybrids failed to produced any shoots. Among the remaining genotypes, two had only sporadic occurrence of shoot formation, two gave an intermediate response (15% and 24% of their discs carried shoots), whereas the discs of the two remaining genotypes responded well to culture (68% and 77%). The genetic analysis performed on the reciprocal hybrids revealed that a positive significant correlation existed between anther induction and leaf disc response (Spearman's r=0.82; p=0.01). This suggests that, under our conditions, these two aspects of tissue culture might share a common system of genetic control. Estimates of broad sense heritabilities, for leaf disc culture, 83% were obtained and the number of effective factors involved in the control of tissue culture response, indicated a relatively simple genetic control. Finally, considering the potentialities opened by the use of RFLP analysis, it might be possible to find probes that are linked with genes involved in tissue culture competence.     

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.     

Wolbachia affects survival to different oxidative stressors dependent upon the genetic background in Drosophila melanogaster

It is well known that the rate of ageing varies among individuals dependent on the genetic background. In the present study, we explore how Wolbachia infection (a common insect endosymbiont bacterium) and oxidative stress interact in ageing with respect to two different genetic backgrounds of Drosophila melanogaster. Naturally infected and cured lines of Drosophila are challenged with either paraquat or l ‐arginine to generate two different types of oxidative stress. We first observe that removing Wolbachia infection shortens the lifespan in one genetic background but not in the other. Wolbachia infection only makes one of the genetic lines more sensitive to paraquat. However, only the line unaffected by Wolbachia in the paraquat treatment is protected by Wolbachia from l ‐arginine induced stress. Hence, Wolbachia is modifying free radical defence via two different mechanisms dependent on the genetic background. This supports the idea that factors that can govern ageing (infection and oxidative stress) are not universal, but are specific to the genetic make‐up of an individual.     

Oxidative stress by antimicrobial peptide pleurocidin triggers apoptosis in Candida albicans

Pleurocidin (GWGSFFKKAAHVGKHVGKAALTHYL-NH₂), found in skin mucous secretions of the winter flounder Pleuronectes americanus, is known to possess a high potency and broad-spectrum antimicrobial peptide without cytotoxicity. In this study, to investigate the impact of pleurocidin on apoptotic progress, we observed morphological and physiological changes in Candida albicans. In cells exposed to pleurocidin, intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased, and hydroxyl radicals were especially a large part of ROS. The increase of ROS induced oxidative stress and mitochondrial membrane depolarization which causes release of pro-apoptotic factors. Using FITC-VAD-FMK staining, we confirmed activation of yeast metacaspases which lead to apoptosis and phosphatidylserine externalization at early stage apoptosis was observed using annexin V FITC. In addition, pleurocidin induced-apoptotic cells underwent apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC) in flow cytometry dot plots. Under the influence of oxidative stress, DNA and nuclei were fragmented and condensed in cells, and they were visualized by 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These apoptotic phenomena represent that oxidative stress by inducing pleurocidin must be an important factor of the apoptotic process in C. albicans.     

Anther culture responsiveness of Hordeum spontaneum derived spring barley lines and a genetic analysis of plant regeneration

Anther culture responsiveness of three H. spontaneum derived spring barley lines, RS170-47(A), RS20-1(B) and 1B-152B(C) was investigated using only one type of culture medium and treatment. The line 1B-152B was identified as highly responsive producing 22.4 total and 12.4 green regenerants per 100 anthers plated. 74% of these green regenerants were spontaneous double haploids. A genetic analysis involving F1 and F2 plants derived from crosses A × B and B × C revealed that the factor(s) determining high anther culture responsiveness in line 1B-152B was heritable and behaved as dominant in the F1. There was an indication that genotypic responsiveness in anther culture for green plant regeneration was different from total or albino plant regeneration.     

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.     

In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

Permanent mycoplasma removal from tissue culture cells: A genetic approach

Mycoplasma contamination of tissue culture cells easily evades detection and, thus, represents a continous threat to cell biologists. In cases where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplasma from the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains; however, cell associated mycoplasmas are often protected from antibiotics at concentrations shown to be effectivein vitro. Antibiotic concentrations high enough to be lethal to cell asso|ciated mycoplasmas frequently are also detrimental to the host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression and/or to the emergence of mycoplasma strains resistant even to high concentrations of the antibiotic applied. Here, a genetic approach for the elimination of mycoplasma from tissue culture cells that overcomes these limitations is described. By expression of a selection marker conferring resistance to an otherwise toxic agent,Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough to be lethal to cell associated mycoplasma while leaving the host cells unharmed. Upon successful mycoplasma eradication, cultivation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained susceptibility to the toxic agent. Cessation of the selection marker expression was shown to result from the loss of the selection marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell chromosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered.     

Physiological characterization of four model Lotus diploid genotypes: L. japonicus (MG20 and Gifu), L. filicaulis, and L. burttii under salt stress

The genus Lotus comprises a heterogeneous group of annual and perennial species. Lotus japonicus (with MG20 and Gifu ecotypes) has been adopted as one of the model legumes in genetic and genomic studies. Other Lotus species, such us Lotus burttii and Lotus filicaulis, have also been used in genetic and genomic studies because of their capacity to produce fertile progenies in crosses with L. japonicus. In the present work, physiological responses to salt stress in four Lotus genotypes were evaluated on the basis of growth and associated parameters, such as photosynthesis, ions, relative water content, oxidative damage and antioxidant system responses, using two NaCl levels applied by acclimation for up to 28 and 60 d. Growth responses varied with plant developmental stage in the four Lotus genotypes. L. japonicus MG20 was found to be a salt-tolerant genotype, mainly when exposed to salt stress at the young plant stage. The capacity of Lj MG20 to sustain growth under salt stress was correlated with enhancement of Superoxide dismutase and Glutathione reductase activities, as well as with increases in total and reduced glutathione content and lower Na⁺ accumulation in leaves. These results suggest that enhancement of antioxidant responses in Lj MG20 contributed to improve salt stress tolerance at early stages. On the other hand, after long-term high NaCl stress treatment, L. filicaulis exhibited lower biomass reduction, lower oxidative damage and Na⁺ accumulation in leaves than the control treatment; hence, this genotype was considered salt-tolerant. These apparently ambiguous results remark that salt tolerance, as a development-related process, was differentially expressed among the Lotus genotypes and depended on stress duration and plant phenological stage.     

Agrobacterium-mediated genetic transformation of the desiccation tolerant resurrection plant Ramonda myconi (L.) Rchb.

In this paper we describe the first procedure for Agrobacterium tumefaciens-mediated genetic transformation of the desiccation tolerant plant Ramonda myconi (L.) Rchb. Previously, we reported the establishment of a reliable and effective tissue culture system based on the integrated optimisation of antioxidant and growth regulator composition and the stabilisation of the pH of the culture media by means of a potassium phosphate buffer. This efficient plant regeneration via callus phase provided a basis for the optimisation of the genetic transformation in R. myconi. For gene delivery, both a standard (method A) and a modified protocol (method B) have been applied. Since the latter has previously resulted in successful transformation of another resurrection plant, Craterostigma plantagineum, an identical protocol was utilized in transformation of R. myconi, as this method may prove general for dicotyledonous resurrection plants. On this basis, physical and biochemical key variables in transformation were evaluated such as mechanical microwounding of plant explants and in vitro preinduction of vir genes. While the physical enhancement of bacterial penetration was proved to be essential for successful genetic transformation of R. myconi, an additional two-fold increase in the transformation frequency was obtained when the above physical and biochemical treatments were applied in combination. All R ₀ and R ₁ transgenic plants were fertile, and no morphological abnormalities were observed on the whole-plant level. Collaborator via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems     

Redox and hormone profiling of a Nicotiana tabacum dedifferentiated protoplast culture suggests a role for a cytokinin and gibberellin in plant totipotency

The ability of plant tissues to retain totipotency despite being fully differentiated has been documented for decades. The transition from mature plant tissue to rejuvenated tissue first requires dedifferentiation of mature tissue, followed by rejuvenation (re-entry into the cell cycle) and somatic embryogenesis. We used a Nicotiana tabacum protoplast-based culture system to elucidate the role played by redox and phytohormone networks during the process of dedifferentiation and rejuvenation. Classical markers of redox homeostasis were measured during the rejuvenation process and lipid peroxidation is proposed as the best marker for indicating recovery of cells from oxidative stress sustained during the process of protoplast preparation and culture, prior to rejuvenation. A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner. A sharp rise at 72 HAC of gibberellin GA₄, furthermore suggests a function for this hormone during the process of rejuvenation. These two key findings are consistent with previously described plant models for lateral root developmental. Therein, iPA could be involved in ‘stem-cell-niche’ initiation. Subsequently, GA₄ could be involved in rapidly suppressing this initiation step following the earliest cell divisions, thereby enabling the establishment of this ‘niche’ into a callus-like tissue.

     

高通量植物生物反应器及其在遗传资源挖掘中的应用

生物反应器(bioreactor)是一种以表达目标产物或获得繁殖体为目的的设备系统,包括微生物、动物、植物生物反应器以及相关设备。植物生物反应器(phytobioreactor)是借鉴植物组织培养和微生物发酵原理制作的设备系统。其中,应用较广泛的是间歇浸没式植物生物反应器。与传统植物组织培养相比,该方法具备可换气、无需转接和大容量培养等特点。国内制作的BIOF系列新型植物生物反应器还可以利用串/并联方法,实现更高通量培养能力,其应用于植物种苗繁育、代谢产物的表达、耐盐等变异的定向筛选、植物生长发育的动态分析等方面均具备显著优势。现代植物生物技术在基础研究和产业方面的应用对植物生物反应器提出了新要求,新型生物反应器应用方法的持续改进和设备系统的不断完善,使其成为植物学领域的高效研究平台,并将促进植物育种和植物源化合物的发掘等方面研究效率的提高。     

An improved,simple, hydroponic method for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis thaliana is one os the most studied plant model systems. Completing the genomic sequence ofA. thaliana has provided new opportunities for physiological and biochemical studies. While its small size is advantageous for genetic studies, the plant's low biomass makes it difficult to obtain enough plant material for biochemical and physiological research. The small size and rosette leaf structure, combined with the sensitivity of the apical meristem to flooding, make hydroponic growth of this model plant difficult. A few systems for hydroponic culture ofArabidopsis have been described. Gibeaut et al. (1997) introduced the use of rockwool forArabidopsis hydroponic culture. We have improved this system by introducing small-volume plastic containers with improved plugs to support the rockwool. This method is simpler than the original setup and provides improved germination and growth. The smaller containers enable the use of this system in growth chambers or small growth rooms for a large number of parallel experiments.