Plant regeneration from thin cell layers in Spinacia oleracea

Caulogenesis and somatic embryogenesis were induced from transverse thin cell layers (tTCLs) of two European (Spinacia oleracea L.) spinach genotypes. Regeneration occurred mostly when tTCLs had been excised from seedlings grown on a preconditioning medium consisting of White's macroelements, Nitsch's microelements, Murashige and Skoog's (MS) vitamins, 6 g l^–1 agar and 20 g l^–1 glucose. The explants were cultured on MS medium supplemented with sucrose (10, 30, 50 or 80 g l^–1) or fructose (5, 10 or 30 g l^–1) and several combinations of indole-3-acetic acid (IAA), -naphtalene acetic acid (NAA), 6-benzylaminopurine (BAP) and gibberellic acid (GA₃). Most of the regeneration events were obtained from root explants of the cultivar Carpo. The best result was observed on MS medium supplemented with 50 g l^–1 sucrose, 100 M NAA, 1 M BAP and 10 M GA₃. After an 8-week culture, the calluses were transferred onto MS medium where shoots and somatic embryos appeared 1 week later. The best root development was obtained on MS medium supplemented with 4.9 M indole-3-butyric acid (IBA) and 8 g l^–1 Phytagel. The plantlets were, then, transferred to soil and developed into well-conformed, fertile plants.     

Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus

 Callus induction, somatic embryogenesis and plant regeneration were obtained in six different citrus species [Citrus deliciosa Ten. (cv 'Avana'), C.limon (L.) Burm. (cv 'Berna'), C.madurensis Lour. (cv 'CNR P9'), C.medica L. (cv 'Cedro di Trabia'), C.tardiva Hort. ex Tan. (cv 'CNR P6'), C.sinensis (L.) Osb. (cv 'Ugdulena 7')] from cultures of pistil transverse thin cell layer explants [(t)TCL]. Explants were cultured on three different media: the nutrients and vitamins of Murashige and Skoog medium alone (MS) or MS supplemented with either 500 mg l^–1 malt extract (MS I) or 500 mg l^–1 malt extract and 13.3 μM 6-benzylaminopurine (MS II). Sucrose (146 mM) was used as the carbon source. Somatic embryos were visible 2–5 months after culture initiation. The different genotypes showed a different embryogenic frequency from stigma, style and ovary (t)TCL explants. All of the cultivars regenerated somatic embryos. Percentages of style (t)TCL explants producing somatic embryos ranged from 0% (C.deliciosa, C.madurensis, C.sinensis and C.tardiva on the three different media) to 5.2% (C.limon on MS II). Embryo formation in stigma (t)TCL explants ranged from 0% (C.madurensis on MS and MS I, C.sinensis on MS, C.deliciosa and C.tardiva on the three different media) to 42.4% (C.limon on MS II). Embryo formation in ovary (t)TCL explants ranged from 0% (C.deliciosa on MS, C.limon, C.medica, and C.sinensis on the three different media) to 9.3% (C.tardiva on MS I). After about 12 weeks somatic embryos developed into plantlets at a high frequency. Received: 22 September 1998 / Revision received: 6 November 1998 / Accepted: 23 November 1998     

Changes in symplastic permeability during adventitious shoot regeneration in tobacco thin cell layers

Thin cell layer (TCL) explants of tobacco (Nicotiana tabacum L.) were cultured in either a regeneration medium that resulted in formation of adventitious vegetative shoots or a non-regeneration (control) medium that maintained the TCLs but did not promote shoot formation. Microinjections were conducted on epidermal cells at 1- or 2-day intervals during the culture period (14 days) and also on meristematic regions as they appeared in regenerating TCLs. A fluorescein isothiocyanate-labelled peptide (F(Glu)₃ MW 799) was used to assess the permeability of the symplast during adventitious shoot regeneration. A period of increased symplastic movement of F(Glu)₃ was detected during day 2 of culture and was significantly greater in regenerating TCLs than in non-regenerating TCLs. This corresponded to the period of the first cell divisions and represents the re-initiation of a meristematic type of symplastic linkage between epidermal cells. A smaller increase in cell-to-cell movement within non-regenerating TCLs indicated a possible stress response as a factor in these changes. Movement of F(Glu)₃ throughout the epidermal symplast of regenerating TCLs returned to pre-culture levels by the time of shoot primordia formation. F(Glu)₃ movement was further down-regulated in non-regenerating TCLs, with a high degree of cell isolation observed. Within newly formed shoots, symplastic movement of F(Glu)₃ cycled between high and low levels.     

High frequency shoot regeneration from Rhynchostylis gigantea (orchidaceae) using thin cell layers

Often regeneration in orchids is only achieved through protocorms, i.e., the juvenile stage. In order to produce directly shoots via bud regeneration both rapidly and with a high frequency, a transverse thin cell layer (tTCL) method is extended to Rhynchostylis gigantea. Transverse thin cell layer (tTCL) explants (0.3--0.5 mm) excised along the stem from the basis to the shoot tip of one-year-old plantlets were cultured on Murashige and Skoog (1962) medium supplemented with different combinations of benzyladenine (BAP), 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ) and 3% sucrose. The optimal combination for maximal bud regeneration was 3 µM BAP and 3 µM TDZ, giving rise to 11.7 buds per tTCL. Roots were obtained with 10 µM forchlofenuron (CPPU) and 1% sucrose. The in vitro plants (> 3 cm long) obtained 4 to 6 weeks after the tTCLs culture were transferred to the greenhouse; their morphology was normal. Efficient micropropagation of direct production of shoots without passing through protocorm stage of orchid species can be achieved using the thin cell layer (TCL) method.     

The role of calmodulin in cell transformation

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca²⁺ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca²⁺ medium at the permissive temperature 34°C while both lines did not replicate at the non-permissive temperature 39°C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca²⁺-sensitivity of growth in the cells going from the permissive to non-permissive temperature.     

Root histogenesis from tobacco thin cell layers

Summary Internode stem expiants ofNicotiana tabacum cv. Samsun, consisting of eight cell layers: epidermis, subepidermal chlorenchyma, collenchyma and cortical parenchyma (i.e., thin cell layers), were cultured under conditions inducing rhizogenesis. The aim was to investigate the histological sequence of adventitious root formation in this system. The earliest cytological events in culture (12 h) were nucleolar extrusions and amitotic nuclear divisions. Though not restricted to a specific cell layer, the two phenomena were more frequent in the subepidermal chlorenchyma, and characterized the first phases (12-96 h) of cell proliferation mainly occurring in this layer. Amitoses were followed by the formation of thin walls within the original cells, resulting in the formation of intracellular clusters. These subepidermal clusters were separated by enlarged cells of the parent tissue, whose nuclei showed nucleolar extrusion. At day 3 the first mitoses were observed in cells having abundant starch inclusions. Amitotic divisions also continued, but less frequently. The increasing frequency of mitoses in the subepidermal chlorenchyma (day 4), as well as in the two underlying collenchymatous layers, contributed to the growth of the superficial clusters, in which small clumps of meristematic cells were formed; these, later (day 9), gave rise to root domes. The 5th cell layer remained undivided for a relatively long time (two weeks). The 6th and 7th layers proliferated mitotically later (from day 8 onwards) than the superficial layers and formed root domes following the same histological sequence. Wound callus, generated by the innermost layer, increased markedly in the last two weeks of culture and concomitantly formed vascular clumps surrounded by meristematic layers; these produced root primordia which were frequently anomalous (day 26–27). Regardless of its origin (i.e., superficial or deep layers of the expiant, or wound callus cells), root tip formation was always preceded by the differentiation of a sheath of starch-containing cells, from which the root cap developed.Abbreviations LS longitudinal section - S.E. standard error - TVS transverse section     

The importance of the explant on regeneration in thin cell layer technology

Summary The basic factor underlying the success of the tissue culture, large-scale micropropagation and genetic transformation of any plant species is regeneration. This has been achieved over the years through the use of various-sized explants ranging from protoplasts (small scale) to entire organs (large scale). Inherent problems underlie the use of either extreme, leading to both nonspecific morphogenic reactions in the latter, or to undesired necrosis in the former. This review investigates the importance of different aspects of a thin cell layer (TCL) explant, from its source to its size. TCLs, as a results of their size and origin, in combination with other controllable factors such as media and environmental conditions, have shown this system to be superior to the use of conventional explants. Numerous species that were previously unsuccessfully tissue-cultured have, with the use of TCL technology, resulted in their successful micropropagation and regeneration. These successes, based on the inherent qualities of the TCL explant—specific for a given species—are also examined.     

Adventitious root formation in Arabidopsis thaliana thin cell layers

This paper describes, for the first time, de novo adventitious root formation from thin cell layers (TCLs) of Arabidopsis thaliana. The objective of the study was to determine the optimal hormonal and light conditions and the optimal exogenous Ca²⁺ concentration for obtaining adventitious rooting (AR) from A. thaliana TCLs and to identify the tissue(s) involved in the process. The results show that maximum AR was obtained with a single-phase method in the presence of 10 M indole-3-butyric acid and 0.1 M kinetin under continuous darkness for 30 days and with 0.6 mM exogenous CaCl₂. The endodermis was the only tissue involved in root meristemoid formation. The role of Ca²⁺ in AR and the importance of using Arabidopsis TCLs in studies on the genetic/biochemical control of AR are discussed.Abbreviations AR Adventitious rooting - CIM Callus-inducing medium - Col-0 Columbia ecotype - 2,4-D 2,4-Dichlorophenoxyacetic acid - HFM Hormone-free medium - HM Medium with 10 M IBA and 0.1 M Kin - IBA Indole-3-butyric acid - Kin Kinetin - LS Longitudinal section - NAA -Naphthaleneacetic acid - RIM Root-inducing medium - TCL Thin cell layer - WS Wassilewskija ecotype     

The role of cell membrane in the regulation of cell division and malignant transformation

An earlier model in which uptake of essential nutrients for which the cell is auxotrophic, regulates cell division, is discussed in the light of new experimental findings, specifically the purification of a new type of transport-inhibitory protein from rat liver and the properties of the protein. The possible role of such proteins in malignant transformation is also discussed.     

Calpain: a role in cell transformation and migration

Calpains represent a well conserved family of calcium-dependent proteolytic enzymes. Recent progress in determining the three-dimensional crystal structure of calpains and generation of calpain knock out animals have significantly advanced our understanding of both the activation mechanism and physiological role of this protease family. Studies applying molecular intervention strategies and genetic ablation of calpain now provide indisputable evidence that calpain activity contributes to remodelling of the actin cytoskeleton, cell migration and oncogenic transformation. Src and epidermal growth factor receptor (EGFR) stimulated cell motility is dependent upon calpain activation. In addition, calpain promotes accelerated cell-cycle progression and anchorage-independent growth of Src transformed cells. In vivo studies demonstrate a link between calpain expression levels and activity with tumour development and invasion. Thus, recent investigations suggest that the role of calpain in promoting cell transformation and cell migration may have important in vivo consequences in the context of cancer pathobiology.     

Production of isolated somatic embryos from sunflower thin cell layers

We describe here a two step procedure which allows the easy isolation of somatic embryos from Sunflower (Helianthus annuus L.) hypocotyl tissues. Thin cell layers composed of the epidermis plus 3 to 6 parenchyma cell layers were incubated for 5 days in a basal Murashige and Skoog medium using an auxin to cytokinin weight ratio of 1/1. The epidermis layers were then transferred to a Gamborg medium containing a high level of sucrose. After one week of incubation in this medium, many somatic embryos started to be released from the parental epidermal tissue. Even though the germination of these embryos is difficult, we have been able to induce secondary embryos and regenerate fertile plants.Abbreviations NAA 1-naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-benzylamino-purine - MS Murashige and Skoog medium - B5 Gamborg medium     

The use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation

Summary Combining stepper-motors for microscope stage movement with a specially designed software program has led to the establishment of an efficient cell tracking system. Cell immobilization, fixed reference points for calibration of target cell positions, and a video recording system complete the cell finder system. Specific cells can be identified (either beforehand or in retrospect), their locations fixed, and subsequent development of the individual cells monitored daily using computer-assisted relocation. In this way, the specific cell type capable of sustained division and regeneration has recently been identified within a low efficiency protoplast system of a recalcitrant species, sugarbeet. These totipotent cells originated from stomatal guard cells. Isolation and purification procedures were then optimized in a directed way to yield millions of guard cell protoplasts (GCPs). Using polyethylene glycol (PEG)-mediated gene transfer and glucuronidase (GUS) activity for transient expression studies proved that GCPs were amenable to transformation. Gene transfer efficiency was high, as was the number of stably transformed plants that can be produced. At present, the optimized procedure yields 600 transgenic individuals per person per year. This number allows for the selection of the best plants with regard to copy number, DNA insert size, gene expression, and field performance. Prospects for future application of the cell finder system will be discussed. Presented as part of the symposium “Early Events in Tissue Culture and Transformation at the Cellular Level” at the 1997 Congress on In Vitro Biology, Washington, DC 14–18 June 1997.     

Organogenesis from hypocotyl thin cell layers of Lupinus mutabilis and Lupinus albus

When thin cell layers (TCLn), isolated from the nodalregion of the hypocotyl of 5 day old seedlings, werecultured in medium M1 with IAA and BA, entire plantswere obtained from L. mutabilis explants andprolific shoots from L. albus explants.Histological studies carried out on L. mutabilisTCLn showed that meristematic cells, present in theexplants at the time of inoculation, gave rise to theformation of a primary meristem resulting inorganogenesis. In TCLn where apical or axillarymeristematic cells were absent, we observed theformation of a nodular structure with a cambium-likelayer at its periphery which divided to form a primarymeristem leading to organogenesis. Plant regenerationfrom TCLn may be useful in genetic transformationassays.     

The role of transformation in the variability of the Neisseria gonorrhoeae cell surface

This review discusses the genetic basis for surface changes in Neisseria gonorrhoeae and the role of specific transformation reactions in producing them. Variation in the structure of pilin, the subunit of gonococcal pili, occurs by transformation-mediated recombination of DNA segments in storage loci with the expression locus. These pilin loci have low recombination potential since their sequences contain only short uninterrupted identical sequences. The DNA within storage or silent loci are also relatively deficient in the short defined sequences which target DNA for efficient uptake and thus have relatively low affinity for the DNA transport system. Consequently, pilin-encoding DNA segments constitute relatively poor substrates for the general transformation system of gonococci. These considerations suggest the existence of locus-specific factors which increase the efficiency of genetic exchange between pilin loci. I raise the speculative hypothesis that one function of transformation-mediated DNA entry is to provide a regulatory stimulus signalling the death of neighbouring gonococci. This regulatory shift might lead to production of factors which accelerate genetic reshuffling of pilin loci either by transformation per se using external DNA as donor, or via a recombinational process which utilizes internally derived DNA segments as donors. A signalling function for transforming DNA also clarifies several general properties of specific transformation reactions.     

The role of epiphytism in architecture and evolutionary constraint within mycorrhizal networks of tropical orchids

Characterizing the architecture of bipartite networks is increasingly used as a framework to study biotic interactions within their ecological context and to assess the extent to which evolutionary constraint shape them. Orchid mycorrhizal symbioses are particularly interesting as they are viewed as more beneficial for plants than for fungi, a situation expected to result in an asymmetry of biological constraint. This study addressed the architecture and phylogenetic constraint in these associations in tropical context. We identified a bipartite network including 73 orchid species and 95 taxonomic units of mycorrhizal fungi across the natural habitats of Reunion Island. Unlike some recent evidence for nestedness in mycorrhizal symbioses, we found a highly modular architecture that largely reflected an ecological barrier between epiphytic and terrestrial subnetworks. By testing for phylogenetic signal, the overall signal was stronger for both partners in the epiphytic subnetwork. Moreover, in the subnetwork of epiphytic angraecoid orchids, the signal in orchid phylogeny was stronger than the signal in fungal phylogeny. Epiphytic associations are therefore more conservative and may co‐evolve more than terrestrial ones. We suggest that such tighter phylogenetic specialization may have been driven by stressful life conditions in the epiphytic niches. In addition to paralleling recent insights into mycorrhizal networks, this study furthermore provides support for epiphytism as a major factor affecting ecological assemblage and evolutionary constraint in tropical mycorrhizal symbioses.     

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01–10 μM MeJA, either under root-inductive conditions, i.e., on medium containing 10 μM indole-3-butyric acid (IBA) and 0.1 μM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1–4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 μM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 μM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.