Cytokinin Habituation Research Articles

Tissue proliferation condition in micropropagated ericaceous plants

Tissue proliferation (TP) is an abnormal tumor-like growth produced at or near the crown of the plant, but may also be found on aerial plant parts of some genotypes. Basal tumors may or may not be accompanied by proliferation of compact shoots with short internodes and a whorled leaf arrangement. Genera exhibiting TP include Rhododendron, Kalmia and Pieris of the Ericaceae family. Development of TP symptoms in a plant is highly correlated to a history of micropropagation and also to genetic background of the genotype. Similarities of TP symptoms to crown gall caused by Agrobacteriumtumefaciens led many to initially believe TP to be a form of crown gall, but all evidence suggests that a pathogen is not involved in the TP disorder. Abnormal lignotuber formation is another possible cause for TP for which little supportive evidence could be developed. An epigenetic condition, possibly cytokinin habituation, is the possible cause of TP that is best supported by the majority of research evidence. It is likely that TP is induced in vitro by adventitious shoot formation, resulting from high cytokinin concentrations used to rapidly proliferate shoots. Some nursery production practices may result in increased TP symptoms development post-propagation. TP is well managed now due to greater awareness and adoption of sound micropropagation practices for ericaceous plants by tissue culture labs.

CYTOKININ HABITUATION FOR AUTONOMOUS SHOOT INITIATION IN PAWPAW (ASIMINA TRILOBA)

Pawpaw is native to Eastern North America and shows good potential as a tree fruit crop. Micropropagation could provide a clonal propagation method for plant production and germplasm preservation. Once established, pawpaw cultures typically produce many shoot-bud clusters that do not readily elongate. Shoot-bud cultures that had been maintained on a BA (8.9 μM) + NAA (2.3 μM) medium for over five years showed evidence of cytokinin habituation. Single shoot-buds (1.5 cm) moved to a media with or without plant growth regulators (PGR) continued to initiate new shoots at a similar rate (∼ 5 to 8 shoots per culture). Shoots on PGR medium failed to elongate shoots greater than 2 cm compared to approximately 12% for those on PGR-free medium. Single shoot-buds subcultured three times to PGR-free medium continued to initiate between 5 to 7 shoot-buds per culture and shoot elongation remained at approximately 16%. This sensitivity to cytokinin and apparent carryover effect may in part explain the recalcitrant nature of pawpaw microcuttings to rooting.

A Transcriptome-Based Characterization of Habituation in Plant Tissue Culture

For the last 50 years, scientists have recognized that varying ratios of the plant hormones cytokinin and auxin induce plant cells to form particular tissues: undifferentiated calli, shoot structures, root structures, or a whole plant. Proliferation of undifferentiated callus tissue, greening, and the formation of shoot structures are all cytokinin-dependent processes. Habituation refers to a naturally occurring phenomenon whereby callus cultures, upon continued passage, lose their requirement for cytokinin. Earlier studies of calli with a higher-than-normal cytokinin content indicate that overproduction of cytokinin by the culture tissues is a possible explanation for this acquired cytokinin independence. A transcriptome-based analysis of a well established habituated Arabidopsis (Arabidopsis thaliana) cell culture line was undertaken, to explore genome-wide expression changes underlying the phenomenon of habituation. Increased levels of expression of the cytokinin receptor CRE1, as well as altered levels of expression of several other genes involved in cytokinin signaling, indicated that naturally acquired deregulation of cytokinin-signaling components could play a previously unrecognized role in habituation. Up-regulation of several cytokinin oxidases, down-regulation of several known cytokinin-inducible genes, and a lack of regulation of the cytokinin synthases indicated that increases in hormone concentration may not be required for habituation. In addition, up-regulation of the homeodomain transcription factor FWA, transposon-related elements, and several DNA- and chromatin-modifying enzymes indicated that epigenetic changes contribute to the acquisition of cytokinin habituation.

Cytokinin Habituation in Juvenile and Flowering Tobacco

Abstract The possible link between cytokinin and flowering was examine in tobacco. The degree of cytokinin autotrophy and the competence for cytokinin habitution were measured in callus derived from pith tissue of Nicotiana tabacum cvs. H425 an W38. Explants were taken from internodes at all positions up the stem in juvenile and mature plants. To test whether the competence of cells to form flowers was linked with crtokinin habituation, thin cell layer explants from comparable internodes were tested for their ability to form floral buds. Callus derived from the upper parts of plants showed cytokinin autotrophy whether or not the plants were flowering. Flower buds were formed only on thin cell layer explants from the upper part of plants which were already flowering. Cytokinin habituation and competence to flower are therefore not directly linked although cytokinin habituation could be a prerequisite for meristematic activity and for flowering. Pith from internodes in the lower half of mature pants formed callus which was cytokinin-dependent, although these same internodes in juvenile plants were cytokinin-autotrophic. The ability to form cytokinin-autotrophic callus was therefore greatest in the meristematic regions and was lost as the pith cells aged. Competence to habituate after 35 °C treatment was also shown by pith callus from a few internodes in the middle of the plant below those already forming cytokinin-autotropic callus.

Suspension culture of sugarbeet (beta vulgaris L.). induction and habituation of dedifferentiated and self-regenerating cell lines

A systematic procedure is described to obtain habituated cell suspensions from different explant sources of sugarbeet (Beta vulgaris L.).Cytokinin habituation occurred spontaneously. Auxin habituation could be induced by successive lowering of the external hormone concentration. Habituation was reversible and independent of the cell type or explant source. The habituated nature of the cells was lost during regeneration and had to be reinduced by adequate treatments.A method for initiating habituated self-regenerating lines was established. These cultures were less stable. Total dedifferentiation caused the loss of the regeneration capacity. Reculturing of the regenerated plants did not result in a higher percentage of primary regeneration.

Reversible, cell-heritable changes during the development of tobacco pith tissues

Cytokinin requiring cells of Nicotiana tabacum L. cv “Havana 425” can be induced in culture to become cytokinin autotrophic. This process is known as cytokinin habituation. Earlier we showed that pith parenchyma tissue consists of inducible cells, which habituate at high rates when treated with cytokinin, and noninducible cells, which remain cytokinin requiring under these conditions. The inducible and noninducible phenotypes are determined states that arise during the development of the tobacco plant and are inherited by individual cells. Here we show that pith tissue of plants regenerated from cloned lines of noninducible cells exhibits the inducible phenotype indicating that noninducible cells, or their descendants, can become inducible. This change in competence for habituation appears to have an epigenetic basis; it is reversible, occurs at high rates, and depends on the developmental state of the cells. The habituated state occurs in two forms that can be distinguished by their difference in developmental potential. Habituated cells derived from inducible pith cells give rise to normal plants whose leaf and pith tissues require cytokinin for growth in culture. In contrast, habituated cells obtained by transferring noninducible cells on media with progressively lower cytokinin concentrations give rise to plants whose leaf and pith tissues exhibit a cytokinin-habituated phenotype in culture.

Cytokinesis, Cell Expansion, and the Potential for Cytokinin-Autonomous Growth in Tobacco Pith

Pith tissue from Nicotiana tabacum L. cv ;Maryland Mammoth' or ;Wisconsin 38' was isolated, free of vascular tissue, and cultured on a medium containing auxin but no cytokinin. Explants from the apical 1 cm of stem, within the pith rib meristem, initiated callus growth with 100% efficiency. Macroscopically visible callus was evident 5 days after the tissue was isolated, and the cultures grew persistently in the absence of cytokinin. Heat treatment, sometimes used to initiate cytokinin habituation, was not required. Explants from tissue basipetal to the pith rib meristem declined in the frequency of habituation with increasing distance from the shoot apex. Although pith tissue which was growing, in vivo, was more prone than mature tissue to establish cytokinin-habituated callus, the basipetal decline in habituation frequency extended well beyond the zone of cell expansion. Explants from mature pith 40 centimeters or more from the shoot apex grew in the absence of cytokinin with 18% frequency, although the response required at least 2 weeks of culture. Further analysis demonstrated that tissue near the periphery of mature pith was more prone to cytokinin-habituation than tissue from the pith center.

Factors influencing the incidence of habituation for cytokinin of tobacco pith tissue in culture

Pith tissue of Nicotiana tabacum L. cv. "Havana 425" exhibits a gradient in its tendency to habituate for cytokinin on an auxin-containing medium at 35° C, about 10° C above the standard culture temperature. Explants of pith from below the 8th to 11th internode, counting from the bottom of the plant, rarely habituate for cytokinin; explants from above this threshold habituate rapidly. The explants must also be above a critical size, about 20-30 mg, to habituate. There was a pronounced interaction between size and position effects; the threshold position for cytokinin habituation shifted upward with decreasing explant size.

Variation in the Competence of Tobacco Pith Cells for Cytokinin-Habituation in Culture

Pith parenchyma tissues of tobacco sometimes lose their exogenous requirement for a cell division factor such as the cytokinin, kinetin. This process, known as cytokinin habituation, appears to involve epigenetic changes since it is a heritable change in cell phenotype which is directed, regularly reversible, and leaves the cell totipotent. In this report, we show that pith cells in culture consist of at least two types of cytokinin-requiring cells. The first type habituates rapidly under inductive conditions. The second type continues to express the cytokinin-requiring phenotype for many cell generations in culture but retains the capacity for habituation. These findings suggest that pith cells differ in their competence to habituate and that different states of competence are inherited by individual cells.

Chromosome number and the degree of cytokinin habituation of cultured tobacco pith cells

Pith cells ofNicotiana tabacum L. cv. “Havana 425” gradually increase in degree of cytokinin habituation when serially propagated in culture. We tested the hypothesis that this variation results from changes in chromosome number. This involved inducing primary expiants of pith tissue to habituate and then analyzing clones derived from the tissues for mean chromosome number and relative growth rates on media with and without the cytokinin, kinetin. The experimental results did not support the working hypothesis. There was no conspicuous correlation between degree of habituation and chromosome number. Chromosome number, karyotypic stability and the capacity of cells for proliferation depended on the manner in which cultures had been initiated. Of particular interest was the observation that clones were predominantly diploid when isolated from pith tissues incubated on an auxincontaining medium at 35 °C.

The induction of cytokinin habituation in primary pith explants of tobacco

Pith parenchyma tissue ofNicotiana tabacum L. cv. "Havana 425" becomes cytokinin habituated when incubated at 35°C on an auxin-containing medium. Under these conditions, habituated, hyperplastic nodules appear on the tissues. We used these nodules to estimate the incidence of habituation by a statistical method. The rate of habituation varied with the season. Tissue isolated from plants in the spring habituated approx. 7 times faster than did tissue isolated from plants in winter. The fact that the average rate, >4×10(-3) per cell generation, was 100-1,000 times faster than the rate of somatic mutation inNicotiana species and depended on the physiological state of the tissue provides further evidence that habituation involves epigenetic changes rather than rare, random genetic mutations. We also found that kinetin (6-furfurylaminopurine) induced habituation and that the concentration required depended on the duration of cytokinin treatment. For long incubation times, approx. 6×10(-10) M kinetin, which is about 1,000-fold lower than the concentration optimal for growth of cytokinin-requiring pith tissue, was sufficient to induce habituation. These results support the hypothesis that the habituated state is maintained by a positive feedback loop in which cytokinins either induce their own synthesis or inhibit their own degradation.

Tissue-Specific Variation in the Cytokinin Habituation of Cultured Tobacco Cells

Cells of tobacco pith parenchyma sometimes lose their requirement for an exogenous supply of a cell division factor usually supplied as the synthetic cytokinin, kinetin. This change in phenotype, known as cytokinin habituation, is inherited by individual cells and appears to result from epigenetic changes rather than from rare, random, genetic mutations. We have found that tissues from different regions of the tobacco plant exhibit different states of habituation in culture. Pith tissues, as reported earlier, are usually cytokinin requiring and rapidly shift to the habituated state in culture. Leaf tissues are very slightly habituated and require kinetin for optimal rates of growth. Tissues from the stem-cortex are initially habituated. Both the leaf and cortex phenotypes are inherited by individual cells and persist for many cell generations in culture. These results show that certain tissue-specific phenotypes persist in culture and provide evidence that a process akin to habituation leading to different stable states of cytokinin requirement occurs in normal development.

Cold-sensitive expression of cytokinin habituation by tobacco pith cells in culture

Cloned, cytokinin-habituated tissues of Nicotiana tobacum L. cv. "Havana 425" are able to grow in culture at 25° C without added cytokinin. These tissues vary in their expression of the habituated phenotype at 16° C. When cytokinin-requiring pith tissues are converted to the habituated state by 35° C treatment, all of the habituated cells are cold sensitive. After several transfers in culture, some of these habituated cells give rise to stable, cold resistant variants. Both phenotypes are inherited by individual cells. Cold sensitive clones at 16° C and non-habituated clones at 16° C as well as 25° C show the same dose response to the cytokinin, kinetin. This suggests that at the physiological level, cold sensitivity results from a decreased production of cell division factors rather than from a decreased affinity of cellular receptors for these factors.

Epigenetic variation of cultured somatic cells: evidence for gradual changes in the requirement for factors promoting cell division.

Cells of higher plant species in culture sometimes lose their requirement for an exogenous supply of a cell division factor that, thereafter, they are able to produce. This heritable change, known as cytokinin habituation, appears to be an epigenetic one rather than a classical mutation because it is directed, potentially reversible, leaves the cell totipotent, and involves the expression of a latent differentiated function. By using cloned cell lines derived from pith parenchyma of tobacco, we have obtained evidence that the habituation process is gradual rather than all-or-none and leads to progressively more autotrophic tissues. Cells in culture show reversible shifts among a range of habituated states but remain totipotent and can be induced to regain their requirement for a cell division factor. Thus, it appears that habituation involves epigenetic changes in a quantitative cellular phenotype. Our findings support the hypothesis that tumor progression in crown gall, a neoplastic disease of higher plants, can be accounted for by heritable changes in the pattern of gene expression.

Is bromodeoxyuridine resistance a consequence of cytokinin habituation in Nicotiana tabacum?

Cytokinin-habituated, as compared to nonhabituated Nicotiana tabacum cv. Havana 425 callus was shown to tolerate higher concentrations of 5-bromodeoxyuridine (BUdR) if cytokinin was added to the medium [Meins, Planta 129, 239-244 (1976)].Our aim was to clarify whether or not BUdR-resistance and cytokinin-habituation are linked characters in callus cultures of our BUdR-resistant tobacco mutant BR 37/21, obtained from Nicotiana tabacum cv. Ottawa.It is demonstrated that BUdR-resistance and cytokinin-habituation are independent characters in the case of this mutant. Non-habituated BR 37/21 callus grows as a resistant line on a medium containing BUdR at a selective concentration (30 mg/l) whereas cytokinin-habituated callus, not selected for BUdR-resistance, behaves as a sensitive line, i.e. turns brown and dies, on the same medium.