Morphological diversity of androgenic carrot plants

The aim of this study was to evaluate phenotypic variation of R0 androgenic plants obtained from four seed sources and donor plants by anther culture. Several morphological traits (leaf size, petiole length, leaf division, cortex colour) and the range of diversity were evaluated. There was large variation in all traits among the donor varieties. Especially leaf division and cortex colour differed significantly among the androgenic plants that came from different seed sources. The plants regenerated from four donor plants of variety 62 were significantly different in most traits except for leaf width and cortex colour. Evaluation of R1 plants will demonstrate whether the R0 variation observed is due to genetic variation or physiological differences from tissue culture.     

Doubled haploid production in Flax (Linum usitatissimum L.)

There is a requirement of haploid and double haploid material and homozygous lines for cell culture studies and breeding in flax. Anther culture is currently the most successful method producing doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this review we focus on tissue and plants regeneration using anther culture, and cultivation of ovaries containing unfertilized ovules. The effect of genotype, physiological status of donor plants, donor material pre-treatment and cultivation conditions for flax anthers and ovaries is discussed here. The process of plant regeneration from anther and ovary derived calli is also in the focus of this review. Attention is paid to the ploidy level of regenerated tissue and to the use of molecular markers for determining of gametic origin of flax plants derived from anther and ovary cultures. Finally, some future prospects on the use of doubled haploids in flax biotechnology are outlined here.     

Multiple patterns of mtDNA reorganization in plants regenerated from different in vitro cultured explants of a single wheat variety

Summary We have previously shown that tissue cultures derived from various explants of the wheat variety Chinese Spring exhibit organ/tissue-specific changes in the organization of their mitochondrial genome. The aim of this work was to study the influence of passage out of in-vitro culture, and subsequent plant regeneration, on the in vitro induced reorganization of this genome. In all cases but one, subgenomic configurations present in both the donor parent and the tissue culture were evident, in corresponding regenerated plants. The presence, in regenerated plants, of subgenomic configurations found in tissue culture but undetectable in the donor parent appeared to be both timeand organ/tissue-dependent. Moreover, when present, these novel organizations were not systematically found in all regenerated plants. Finally, novel subgenomic configurations were specifically detected after passage out of in-vitro culture. As these results were obtained from a single plant variety, they clearly confirm the extreme plasticity of mitochondrial genome structure in response to in-vitro culture.Accepted by D. R. Pring     

Does differential seed siring success change over time or with pollination history in wild radish, Raphanus sativus (Brassicaceae)?

Previous work with wild radish has shown that pollen donors sire different numbers of seeds and that the condition of the maternal tissue affects seed paternity, suggesting that both pollen donor characteristics and maternal tissue affect mating. However, because these results are from the greenhouse, it is difficult to know whether they would hold true in the field. Here, we performed hundreds of crosses on several maternal plants to simulate changes during the flowering season of field plants. During the experiment, maternal resource availability changed due to the costs of producing fruits, and we determined the pollination history of a plant by performing crosses in specific orders. Examination of seed paternity showed that there were small differences in pollen donor success at the beginning of the experiment when maternal resources were abundant. Differential pollen donor success was greatest slightly later in the flowering period, but declined toward the end of the experiment. Thus, maternal plants may distinguish most among pollen donors when they have both abundant resources and experience with the differences in quality of available pollen donors. In contrast, there were few significant effects of the recent pollination history of plants on pollen donor success. Finally, despite the changes in mating performance over time, there were strong overall differences in pollen donor success, suggesting that seasonal changes in the field will not eliminate the potential for nonrandom mating.     

Genetic instability during embryogenic cloning of celery

Genetically marked tissues of celery (Apium graveolens) were employed to contrast genetic and chromosomal stability in serially bulk-transferred callus and regenerated plants. After six months in culture, 84% of the callus cells were karologically indistinguishable from normal, while the remainder exhibited chromosome loss and/or fusion. All of 50 clones derived from this tissue expressed the control phenotype with respect to heterozygous isozyme markers. Of 95 plants regenerated from the same tissue, 94 were phenotypically indistinguishable from the original explant donor, and cytogenetic analyses revealed the presence in 4.3% of an accessory chromosome, while the remainder were normal diploids. Analysis of the selfed progeny of these regenerated plants revealed the presence of a new recessive mutation causing abnormal leaf morphology at a frequency of 1.8%. Only one of 40 cells in 12-month-old callus tissue was karyologically indistinguishable from normal, the remainder consisting primarily of hypodiploids. The observation that all 50 clones were phenotypically heterozygous was statistically inconsistent with the hypothesis that hypodiploidy was associated with random complete chromosome loss. The culture had, at this point, lost the ability to regenerate. It is speculated that embryogenic cloning of celery may be suitable under certain circumstances for direct field establishment, but that levels of new genetic variation are sufficiently high to preclude its use for seed production.     

Quantification of the tissue-culture induced variation in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background  

When plant tissue is passaged through in vitro culture, many regenerated plants appear to be no longer clonal copies of their donor genotype. Among the factors that affect this so-called tissue culture induced variation are explant genotype, explant tissue origin, medium composition, and the length of time in culture. Variation is understood to be generated via a combination of genetic and/or epigenetic changes. A lack of any phenotypic variation between regenerants does not necessarily imply a concomitant lack of genetic (or epigenetic) change, and it is therefore of interest to assay the outcomes of tissue culture at the genotypic level.     

Accelerated production and identification of fertile,homozygous transgenic wheat lines by anther culture

Anther culture has been developed in the winter wheat cultivar Florida to achieve accelerated production and identification of homozygous transgenic lines. With untransformed, seed-derived plants to develop the culture system, it was shown that cold pre-treatment of spikes excised from donor plants and addition of 2,4-dichlorophenoxyacetic acid together with either kinetin or 6-benzylaminopurine in the callus induction medium improves the anther culture response. The procedure developed allowed production of fertile homozygous lines within 8–9 months, which includes an 8-week vernalisation period. With transgenic wheat plants produced by particle bombardment as donors, we show that the system can be used to produce homozygous transgenics, requiring one generation cycle. Both T₀ tissue culture-derived plants and their T₁ seed-derived descendents serve as suitable donors. We show that an anther culture response comparable to that of untransformed, seed-derived plants can be achieved with T₀ tissue culture-derived plants. PCR and Southern molecular analyses of anther culture-derived transgenics show that the transgenes are stably inherited; there are no perturbations at the chromosomal level around the sites of transgene integration as a result of in vitro chromosome manipulation during anther culture.     

提高光敏感雄性不育水稻花粉单倍体育种效率及不育花粉植株育性转换特点的研究

比较了(光敏s/正常品种)F_1及F_2为供体亲本,对在花药培养时所获得的花粉植株中不育个体/全部花粉植株之比例的影响。结果表明,以F_1为供体亲本,在所获得的二倍体花粉植株(A_1)中,不育株(长日下)约占20%左右;而从F_2分离的不育株为供体亲本,相应的比例为90%左右。对获得不育的花粉植株而言,供体亲本经过F_2的选择,在花粉一代中可以提高育种效率3—4倍。指出,以培育光敏感雄性不育系为目的的花药培养,与一般育种之花药培养采用杂种F_1为供体亲本不同,不仅应对杂种F_2代在长日照条件下进行不育株的选择,而且应在短日照下对这种不育株作育性转换的双重选择。以这种个体作为花药培养的供体亲本,可以大大提高育种效率。 在长日照下表现不育的花粉植株的育性转换具多样性。来自同一组合的不育花粉植株在晚造(短日照)条件下,其花粉有的染色,频率高且稳定;有的虽然可变为染色,但频率不高或不稳定或二者兼有;有些却一直不为Ⅰ-KⅠ染色,或即使染色频率也在10%以下。这一结果与收集全国各地15个光敏核不育系在本昕同期种值条件下的反应十分吻合。这说明通过花药培养,从特定的组合培育出所需要的光敏/光温互作或温敏型的核不育系的可能性是存在的。     

Interactions of donor sources and media influence the histo‐morphological quality of full‐thickness skin models

Human artificial skin models are increasingly employed as non‐animal test platforms for research and medical purposes. However, the overall histopathological quality of such models may vary significantly. Therefore, the effects of manufacturing protocols and donor sources on the quality of skin models built‐up from fibroblasts and keratinocytes derived from juvenile foreskins is studied. Histo‐morphological parameters such as epidermal thickness, number of epidermal cell layers, dermal thickness, dermo‐epidermal adhesion and absence of cellular nuclei in the corneal layer are obtained and scored accordingly. In total, 144 full‐thickness skin models derived from 16 different donors, built‐up in triplicates using three different culture conditions were successfully generated. In univariate analysis both media and donor age affected the quality of skin models significantly. Both parameters remained statistically significant in multivariate analyses. Performing general linear model analyses we could show that individual medium‐donor‐interactions influence the quality. These observations suggest that the optimal choice of media may differ from donor to donor and coincides with findings where significant inter‐individual variations of growth rates in keratinocytes and fibroblasts have been described. Thus, the consideration of individual medium‐donor‐interactions may improve the overall quality of human organ models thereby forming a reproducible test platform for sophisticated clinical research.     

Microbial hazards in plant tissue and cell cultures

Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.     

A simple plant and tissue culture growth chamber for the regeneration of tobacco mesophyll protoplasts

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.     

Culture of Cotyledon and Hypocotyl Protoplasts from True Potato Seedlings in Solanum tuberosum L.

Culture of protoplast using cotyledon and hypocotyl as the donor tissue from true potato seedlings (TPSs) of 3 breeding lines (DTO-33, ND 860-2 and BN 9815-3) of Solanum tuberosum L. was studied. The cotyledons and hypocotyls of TPSs just extended were excised and digested in an enzyme solution containing 1 % cellulase and 0. 5 % macerozyme for 17—20 h after vacuum infiltration of the tissue in the solution. The protoplasts were cultured in an improved liquid medium and transferred onto solid media for callus culture and shoot regeneration. Some factors affecting the efficiency of cotyledon and hypocotyl protoplast culture were studied. The results showed that using the cotyledons and hypocotyls as donor tissues for protoplast isolation and culture in potato, the division frequency of protoplast derived cells was significantly higher than that using the leaves and shoot-tips of the test-tube plantlets: the yield and quality of the protoplast from TPSs cultured under continuous high light intensity (3000 Ix) were much higher than the TPSs cultured under low light intensity (1000 Ix), and no intact protoplast was ever obtained from the TPSs cultured in continuous dark condition. Vacuum infiltration of the cotyledon and hypocotyl segments in enzyme solution before digestion increased protoplast yield. The yield of protoplasts from hypocotyl tissue was significantly higher than from the cotyledon, but there was no significant difference in quality between the protoplast derived from the two tissues. The significance, advantages and shortcomings of using the cotyledons and hypocotyls as the donor tissues for isolation and culture of potato protoplasts are dicussed.     

Fabrication, quality assurance, and assessment of cultured skin substitutes for treatment of skin wounds

Advances in treatment of skin wounds depend on demonstration of reduced morbidity or mortality either during or after hospitalization. Tissue engineering of skin grafts from cultured cells and biopolymers permits greater amounts of grafts from less donor tissue than conventional procedures. Autologous keratinocytes and fibroblasts isolated from epidermis and dermis of skin may be combined with collagen-based substrates to generate cultured skin substitutes (CSS) with epidermal and dermal components. By regulation of culture conditions, CSS form epidermal barrier and basement membrane, and release angiogenic factors that stimulate vascularization. Prototypes of CSS may be tested for safety and efficacy by grafting to athymic mice which do not reject human tissues. Clinical application of CSS requires establishment of quality assurance assessments, such as, epidermal barrier by measurement of surface hydration, and anatomy by standard histology. Medical benefits of tissue engineered skin for treatment of burns are evaluated quantitatively by the ratio of healed skin to donor skin, and qualitatively by the Vancouver Scar Scale. These benefits may also be extended to other medical conditions including chronic wounds and reconstructive surgery.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

Response to NaCl of alfalfa plants regenerated from non-saline callus cultures

Salinity restricts crop productivity in many arid environments. Inadvertent selection for tolerance to osmotic stress may occur under cell or tissue culture conditions and could affect the performance of regenerated plants. The effect of NaCl on forage produced by alfalfa (Medicago sativa L.) plants regenerated from non-saline callus cultures was examined in this study. Plants of Regen-S, which was selected for improved callus growth and regeneration in non-saline cultures, had higher forage weight when grown on SHII medium at NaCl levels up to 100 mM compared to its parental cultivars, Saranac and DuPuits. Five additional original-regenerant plant pairs, each derived from non-saline callus cultures of different alfalfa plants, were evaluated in a solid (soil-like) substrate under saline and non-saline conditions. Weight of forage produced by rooted stem cuttings of regenerated plants was 33% higher at 50 mM NaCl compared to cuttings of explant donor plants. Self progenies from four of five regenerants had higher relative forage weight at 100 mM NaCl (percent of 0 NaCl treatment) than the original plants indicating increased NaCl tolerance.     

Endogenous IAA and morphogenesis in tobacco petiole cultures

Short duration (48 h) dark or high light treatment of the donor plants has been shown to influence the pattern of auxin metabolism in tobacco petioles in culture. In explants from dark treated donor plants there was a peak of IAA detectable at day 3 in culture. This was associated with reduced morphogenetic potential of the explant. Inclusion of 2,3,5-triiodobenzoic acid in the medium prevented this IAA accumulation and eliminated the inhibitory effect of the dark pretreatment. Inclusion of IAA in the culture medium reduced the morphogenetic potential of explants from high light treated donor plants but had no effect on the morphogenetie potential of explants from dark treated donor plants. Explants cultured for one day on auxin-free medium and then transferred to IAA-containing medium were sensitive to auxin; those transferred after five days were insensitive. Studies on the interaction between media sucrose concentrations, endogenous IAA and peroxidase, and morphogenesis are reported. The results are discussed in relation to the influence of endogenous auxin on the receptivity of explants to exogenous (media) morphogenetic stimuli.     

Transfer ribonucleic Acid modification and its relationship to tumorous and nontumorous plant growth

Detailed analyses of tRNA hydrolysates from four tissue types of Nicotiana tabacum, pith from intact plants, pith growing in culture, habituated tissue in culture, and crown gall tumor tissue in culture, revealed significant qualitative and quantitative differences in the pattern of methylation. Although pith from intact plants and pith growing in culture possessed seven different methylated nucleosides, only two were found in habituated and tumorous tissues in culture. Four of the five compounds accounting for the difference were tentatively identified as methylated guanosines. Evaluation of results in terms of several parameters, including growth rate, the tumorous state, habituation, tissue culture, and potential for differentiation, indicate that the extent of tRNA methylation may be correlated with the potential for differentiation of a particular tissue.     

Report of the clinical donor case workshop of the European Association of Tissue Banks annual meeting 2014

The European Association of Tissue Banks (EATB) donor case workshop is a forum held within the program of the EATB annual congress. The workshop offers an opportunity to discuss and evaluate possible approaches taken to challenging situations regarding donor selection. Donor case workshops actively engage participants with diverging background and experience in an informal, secure and enjoyable setting. The resulting discussion with peers promotes consensus development in deciding tissue donor acceptability, especially when donor health issues are not conclusively addressed in standards and regulations. Finally the workshop serves to strengthen the professional tissue banking networks across Europe and beyond. This report reflects some of the discussion at the workshop during the annual congress in Lund, Sweden, in 2014. The cases presented demonstrate that the implications of various donor illnesses, physical findings and behaviours on the safety of tissue transplantation, may be interpreted in a different way by medical directors and other professionals of different tissue facilities. This will also result in diverging preventive measures and decisions taken by the tissue facilities. Some of the donor cases illustrate varied responses from participants and demonstrate that operating procedures, regulations and standards cannot comprehensively cover all tissue donor illnesses, medical histories and circumstances surrounding the cause of death. For many of the issues raised, there is a lack of published scientific evidence. In those cases, tissue bank medical director judgement is critical to guarantee transplantation safety. This judgement should be based on a proper and documented risk assessment case by case. Conditions or parameters taken into account for risk assessment are amongst others, the type of tissue, the type of processing, the characteristics of the final product, and the availability of an adequate sterilisation methodology. By publishing these difficult donor suitability cases, and the resulting discussions, we provide information for future similar cases and we identify needs for future literature review and scientific research. In this way the donor case workshops play a role in optimizing the quality and security of tissue donation.     

Donor Plant Growth Factors Affecting Anther Culture of Maize and Sweetcorn (Zea mays)

Anthers from seven unselected commercial sweetcorn lines andten experimental maize lines were cultured on a liquid/solidbi-layer culture medium, containing 13 % maltose as the carbonsource. Mean anther efficiencies (number of embryos per 100anthers plated) of 0 to 27.6 % were recorded, with the maximumefficiency of 57.1 % from one plant. The anther efficiency wasfound to be dependent on genotype, microspore developmentalstage and the growth temperatures of donor plants. Immaturemicrospores were found to continue their development duringthe cold pretreatment of the spikes, and this in turn influencedthe level of response to culture. Direct regeneration of embryoidsto plants occurred most frequently when well formed uni- orbi-polar embryos were produced. The quality of embryo producedwas apparently inversely correlated with the number of embryosproduced. Zea mays, haploid culture, embryos, microspores     

Androgenic response of genotypes selected from Capsicum annuum L.?×?C. chinense Jacq. hybrids

In research on androhaploids in the progeny of interspecific hybrids within the Capsicum genus, three genetically stable lines of F₇ generation, selected from the C. annuum L. × C. chinense Jacq. hybrid, were used. In the first line, only callus tissue was formed as a reaction to the conditions of culture. Cytometric analysis of this tissue revealed the presence of cells with DNA content in nuclei at the level of 1C to 16C. The tissue was mixoploid and non-embryogenic. Anthers of the other line did not respond. In the third one, nine embryos were obtained, and they developed into plants. By means of cytometric analysis, the 1C DNA level was found in eight of them and these were androgenic plants. The origin of one of the diploid plant was not established due to the homozygous character of the donor plants. The experiment results confirm the already known diversity of genotype reaction to the conditions of culture. It moreover point to the possibility of selection of the forms with an androgenic potency from interspecific hybrids.