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.     

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.     

A cytokinin mutant derived from cultured tobacco cells

Tissues cultured from the leaf lamina of wild-type Nicotiana tabacum L. cv. "Havana 425" plants require an exogenous source of cytokinin for rapid growth. In contrast, leaf tissues of plants heterozygous or homozygous for the partially dominant, monogenic habituated leaf (H1-1) trait, exhibit a cytokinin-autotrophic phenotype in culture. Here we show that the H1 trait can arise in culture. Cytokinin autotrophic variants were obtained by culturing wild-type tissues of leaf lamina successively on media containing reduced concentrations of the cytokinin, kinetin. Plants regenerated from clones of these variants exhibited the H1 phenotype, which segregated in breeding tests as expected for a dominant, monogenic trait. This trait, designated H1-2, is inherited at a different locus than the H1-1 trait described earlier. Our results show that cytokinin mutants can arise in cell culture and that at least two genes regulate the cytokinin requirement of cultured tobacco tissues.     

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.Abbreviations CDF Cell division factor(s) - NAA -naphthalencacetic acid     

Nuclear DNA content and phytohormone requirements of normal, crown gall and habituated tissues of Nicotiana tabacum var. White Burley

Determination of the nuclear DNA content of leaves and normal, habituated and Crown gall callus tissues of Nicotiana tabacum var. White Burley were performed using cytophotometry on Feulgen stained preparations. Several aspects concerning the reliability of the Feulgen technique for DNA determinations were investigated.Crown gall callus tissue used in this study had both a higher nuclear DNA content and chromosome number than normal callus (3.2C versus 2.5C). Both have a higher DNA content than the diploid tobacco leaf cells (2C).The normal callus tissue failed to grow on medium without indole acetic acid and kinetin when cultured in tubes. From this normal callus two habituated lines growing without both phytohormones were selected by culturing the normal callus first in the absence of either indole acetic acid or kinetin. Changing the culture conditions of the normal callus by using culture flasks instead of tubes resulted in a remarkably faster growth rate of the tissue. This was accompanied by an acquisition of the habituation characteristics since it was possible now to grow this tissue also directly on medium lacking both phytohormones. All habituated tissues showed a higher nuclear DNA content compared to the normal callus tissue from which they were derived. Interestingly, one of the tissues acquired a nuclear DNA content not different from that of Crown gall tissue. By changing the culture conditions of Crown gall callus tissue no concomitant change in nuclear DNA content occurred.The results suggest a correlation between the acquisition of a special chromosome complement and the loss of phytohormone requirement resulting in autonomous growth.     

Transdetermination of plant cells

Tissues derived from the leaf lamina of Nicotiana tabacum L. cv. Havana 425 plants require cytokinin for continuous proliferation in culture, whereas tissues derived from the cortex of the stem are cytokinin autotrophic. Both phenotypes persist when the two types of cells are cloned, indicating that leaf- and cortex-derived cells are determined to express different cytokinin requirements in culture. We showed that cultures derived from leaf and cortex tissues of plants regenerated from cloned leaf and cortex cells exhibit the cytokinin requirement of comparable tissues of seed-grown plants. This provides direct evidence that plant cells can undergo transdetermination and that this process has an epigenetic basis.     

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.     

Evidence for a Mendelian factor controlling the cytokinin requirement of cultured tobacco cells

Cultured leaf tissues of Nicotiana tabacum L. cv. “Havana 425” normally require an exogenous source of cytokinin for rapid growth; stem-cortex tissues do not—ie, they exhibit the cytokinin-habituated phenotype. We found that plants regenerated from cloned cortex and leaf tissues from one particular plant differed in leaf-tissue phenotype: Leaf tissues derived from leaf cells exhibited the normal, nonhabituated phenotype, whereas leaf tissues derived from cortex cells were cytokinin-habituated. This difference in leaf phenotype was not found using leaf and cortex cells from six other donor plants. The inheritance of the habituated leaf trait was studied in tissues from cortex-derived plants and hybrids between these plants and normal plants. F₁ hybrids were intermediate between the parental types in degree of habituation. No differences were found between reciprocal hybrids. These results suggest that the habituated leaf trait is an incompletely dominant, nuclear trait. Both parental and intermediate phenotypes were recovered in the F₂ progeny. The frequency of habituated leaf progeny in the F₂ and backcross populations provide evidence that the trait is regulated at a single genetic locus.     

Rapid Reversion of Cell-Division-Factor Habituated Cells in Culture

Abstract. The heritable conversion of cell-division factor (CDF)-requiring tobacco pith cells to the CDF-autotrophic state, known as CDF habituation, is a directed process that occurs at very high rates in culture. Here we show that tiny, leafy buds arising from habituated tissues placed on an inductive medium exhibit the non-habituated phenotype. The appearance of these buds was used to estimate the rate at which habituated cells revert. The rate obtained, approx. 4 × 10^-3 per cell generation, is at least 100 to 1,000 fold higher than expected for somatic mutation. Moreover, reversion is a directed process, i.e., it occurs at high rates only under conditions that promote bud formation. These findings provide compelling evidence that habituation of tobacco pith cells involves epigenetic changes rather than rare, random genetic mutations.     

Cytokinin autotrophy and differentiation in tissue cultures of haploid Nicotiana tabacum L.

Explants from the apical region (10 cm from the tip) of haploid Nicotiana tabacum cv. Wisconsin-38 were cultured on media with and without kinetin. Cell lines were selected in the dark and in the light. Cytokinins were extracted from the apical region of haploid plants and from callus tissues after 84 days of growth (third transfer culture). Chlorophyll was extracted from callus grown under light after 21 days of growth at each of the four cell line selection steps. Kinetin (+) cell lines and cytokinin autotrophic tissues grown in the light showed a compact growth pattern. Microscopic examination of these callus showed the presence of large numbers of nodules consisting of tracheary elements, parenchymatic cells, sieve elements and meristematic cells. Cytokinin-autotrophic callus grown in the dark showed an irregular growth pattern presenting regions of compact tissue and friable tissue. The compact tissue contained large amounts of nodules similar to those of kinetin (+) tissues and of cytokinin autotrophic tissues grown in the light. Extraction of the compact and the friable callus components showed high cytokinin activity in the compact region and low activity in the friable portion. It is suggested that cytokinin synthesis is related to the differentiation of the nodular structures. The amount of chlorophyll increased during the process of cytokinin autotrophic cell line selection.     

Origin of cytokinin-and auxin-autonomy and changes in specific proteins in tobacco callus tissue

On the basis of earlier data it was suggested that the induction of cytokinin autonomy might be accompanied by disorders in plastid function and a decrease in cytokinin utilization. In the work presented below the formation of chlorophyll and the isozyme patterns of nine enzymes, some of which are known to be localized in plastids, were compared in tobacco callus tissues differing in their hormonal requirements. Tissues either not requiring cytokinin or both auxin and cytokinin for their growth, contained a lower amount of chlorophyll than the cytokinin-and auxin-dependent strain. The number of isozymes of glucose-6-phosphate and NADP-malate dehydrogenase (i.e. enzymes which are known to be located in plastids) was reduced from four in the cytokinin-and auxin-dependent strain to two and one in the two cytokinin-autonomous strains, respectively. The fully habituated tissue contained an additional isozyme of NADP-malate dehydrogenase. The total number of isozymes of the remaining enzymes (NAD-malate dehydrogenase, peroxidase, esterase and a-and β-galactosidase) either was decreased or not changed in the cytokinin autonomous strains. The exception was an additional anodic peroxidase in one strain. The number of these isozymes in tissue habituated with respect to both auxin and cytokinin either remained the same or increased. Tobacco callus strains with altered requirements for growth regulators contained some new isozymes which were not present in any other strain and some isozymes present in other strains were absent. These differences are discussed in relation to the possible role of plastid function disorder associated with habituation.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

Hormonal control of 5-bromodeoxyuridine (BUdR) tolerance in crown-gall tumors and habituated tissues

We have found that the unorganized tobacco crown-gall tumor, isolated in our laboratory, and Braun's teratomatic tumor both exhibit the capacity to grow in the presence of high toxic concentrations of BUdR (up to 10^-3 mol·l^-1). Double cytokinin-auxin habituated tissues also exhibit a constitutive BUdR-tolerance. In cytokinin-habituated tissues the BUdR-tolerance seems to depend on the degree of cytokinin autonomy. The presence of exogenous cytokinin significantly increases BUdR-tolerance, and the presence of BUdR suppresses the inhibitory effect of exogenous cytokinin upon habituated tissues. The results clearly show that the disposition of a cell for transient BUdR tolerance requires at least active expression of genes for cytokinin synthesis and that the tolerance becomes permanent when the auxin system is turned on. We conclude that BUdR-tolerance is connected with the establishment of a certain hormonal regulator pattern, depending on the state of cellular differentiation.     

CELL DIVISION IN RELATION TO CYTODIFFERENTIATION IN CULTURED PEA ROOT SEGMENTS

One mm-thick segments cut 10–11 mm proximal to the root tip of germinating seeds of garden pea Pisum sativum were cultured in sterile nutrient medium containing auxin in the presence and absence of kinetin. In the absence of added cytokinin, pericyclic proliferation occurred, the cortical tissues showed no proliferation and were sloughed off, and a callus tissue of diploid cells was formed. In the presence of kinetin concentrations from 0.1–1.0 ppm cortical cells of the segments were induced to divide, beginning at the third day. From experiments with ³H-thymidine incorporation at different times of culture, from cytological squash preparations and from histological sections it was shown that the cortical cells stimulated to divide by cytokinin underwent DNA synthesis prior to division, were polyploid, and following cell division rapidly underwent cytodifferentiation at 5–7 days to form mature tracheary elements. At 10 days, when over 300,000 new cells had been formed per segment about 16% of these cells had formed tracheary elements. It was concluded that cytokinin, together with auxin, was essential for the initiation of DNA synthesis in the cortical cells, for their subsequent division, and finally for their specific cytodifferentiation.     

Cytokinin Habituation in Juvenile and Flowering Tobacco

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.     

Evidence for an abundant 33,000-dalton polypeptide regulated by cytokinins in cultured tobacco tissues

When cloned pith and leaf tissues of Nicotiana tabacum L. cv. Havana 425 are subcultured for 3 d on auxin-containing medium and labelled for 18 h with [³⁵S]methionine, up to 10% of the labelled, soluble-protein fraction is found in a single band with an apparent molecular weight of approx. 32,000–34,000 dalton on sodium-dodecylsulfate polyacrylamide-gel electrophoretograms. The labelling of this band, designated P33, is dramatically inhibited by the cytokinin, kinetin, in some cell lines at concentrations as low as 1.4·10^-8 M. P33 is a major component of the protein fraction obtained from non-habituated clones, cytokinin-habituated clones, and revertant subclones of crown-gall-transformed clones, but cannot be detected in clones habituated for both auxin and cytokinin, or crown-gall-transformed clones. The evidence supports the hypothesis that cytokinin in the presence of auxin regulates the production of a specific, major polypeptide in the soluble-protein fraction of the tissue and that this protein is not produced in tissues autotrophic for both auxin and cytokinin.     

The role of hormones on morphogenesis of thin layer explants from normal and transgenic tobacco plants

The organogenic potential of thin layer stem explants of non-reproductive tobacco plants was tested on a hormone-free medium and under various hormonal conditions. A comparison was made between thin layers excised from normal and transgenic plants at the same developmental stage. The transgenic plants were transformed by insertion of TR- and TL-DNA from Agrobacterium rhizogenes 1855 root-inducing plasmid. The aim was to identify hormonal conditions capable of stimulating the expression of the flowering competence present in the differentiated stem tissues at the induced stage before any visible sign of transition to reproductive development. Flower neoformation, observed at the end of the culture period (day 25), occurred on untransformed thin layers only with kinetin treatment. Explants from transgenic plants showed flower bud regeneration on hormone-free medium, indoleacetic acid alone (1 μ M ), kinetin alone (1 μ M ), and most abundantly on indoleacetic acid plus kinetin (1 μ M each). No flower formation was observed on indolebutyric acid plus kinetin (10 μ M and 0.1 μ M , respectively) in both normal and transgenic explants. The latter treatment enhanced rooting instead, above all in the transgenic explants. On hormone-free medium vegetative bud formation was well expressed both by untransformed and transgenic explants, and enhanced by the combined, equimolar concentrations of indoleacetic acid and kinetin.
The results show that cytokinin allows flowering in florally determined stem explants from normal plants. In the transgenic explants, the flowering response increases when indoleacetic acid is added to cytokinin, thus suggesting a role for auxin in enhancing the expression of the florally determined state in thin cell layers of non-reproductive plants.     

Solasodine Yield in Callus Cultures of Solanum Iaciniatum Ait. Selected for Growth on Hormone-free Medium

Callus autotrophic for both auxin and cytokinin was selectedfrom a hormone-requiring culture of Solanum laciniatum Ait.The control of solasodine yield, callus growth and adventitiousshoot initiation by some exogenous growth regulators and/orlight in the habituated culture was determined. Solasodine concentrationvaried from 0.3 to 1.4 mg g^–1 d.wt. Solanum laciniatum, callus, habituation, solasodine     

Cytokinin effects on growth of quiescent tobacco pith cells

Excised pith tissue from Nicotiana glauca or the tumor-prone hybrid N. glauca × N. langsdorffii has no growth requirement for exogenous cytokinins. Addition of kinetin to cultures of these lines results in growth inhibition at a kinetin concentration 1000-fold lower than the optimal level for kinetin-requiring lines. Cytological comparison of the kinetin-inhibited 2N hybrid and glauca tissues with pith from the kinetin-requiring N. tabacum var. Wisconsin 38 suggests that the nature of the cytokinin action is similar in both situations and that the primary function of cytokinin, when it stimulates growth, may be to curtail cell expansion, thereby facilitating a balance of cell expansion and division requisite for maximal growth.