Tracheid Differentiation Research Articles

Hormones and metabolites which control tracheid differentiation, with or without concomitant effects on growth, in cultured tuber tissue of Helianthus tuberosus L.

When tuber slices of Helianthus tuberosus, are cultured in liquid media containing minerals, thiamine, 2% sucrose, and 1 mg/l α-naphthalene acetic acid (NAA) for 14 days, 3-5% of the cells differentiate into tracheids. Addition of benzylaminopurine (BAP) at 5 mg/l, and 4% starch, which act as xylogenic stimuli, has little effect on growth but results in a yield of over 30% tracheids in the same time period. 70-80% of the tracheids appear between days 8 and 14 of culture. Addition to optimal tracheid media of gibberellic acid (GA3) or abscisic acid (ABA) inhibits tracheid differentiation by 80-90%. GA3 causes a concomitant 20-40% reduction in total cell number while ABA has no effect on growth. In media containing only NAA (lacking BAP and starch), ABA has a large stimulatory effect on the rate of cell division, producing 50% more cells than controls. Sucrose and glucose are equally effective sugars for differentiation, but use of the two in combination results in an unusual interaction leading to substantial inhibition of tracheid formation. There is some indication that the high level of BAP required in tracheid differentiation is acting only at the first regulatory step controlling formation of provascular cells, and not in later steps of cellular development.

INDUCTION OF XYLOGENESIS IN PITH PARENCHYMA EXPLANTS OF LACTUCA

Explants of pith parenchyma excised from Romaine lettuce heads (Lactuca sativa Linn. var. Romàna) exhibited xylogenesis after four days dark incubation on a Murashige and Skoog (1962) medium containing auxin (IAA, NAA, or 2,4‐D), cytokinin (zeatin, kinetin, or benzyladenine), sucrose, and agar. With the exception of 2,4‐D, xylogenesis required both an exogenous auxir and cytokinin. The greatest numbers of tracheids were produced by the IAA (5.0 mg/liter)‐zeati (0.1 mg/liter) and 2,4‐D (0.07 mg/liter)‐zeatin (0.1 mg/liter) treatments, whereas the most effective treatment for callus formation was the NAA (0.5 mg/liter)‐zeatin (0.1 mg/liter) medium. In the absence of exogenous cytokinin, 2,4‐D stimulated xylogenesis after 14 days culture at a concentration of 0.02 mg/liter, but was ineffective at 0.07 mg/liter and 0.2 mg/liter. The 2,4‐D treatment induced tracheids to form in small meristematic nodules; aberrant tracheids were also observed. Different patterns in the differentiation of tracheids were associated with the various auxin‐eytokinin treatments. All IAA‐cytokinin treatments produced perpendicular strands around the periphery and tracheid formation throughout the lower half of the explants. The pattern of IAA‐induced xylogenesis was modified by the particular cytokinin employed. Treatment with NAA‐cytokinin induced horizontal strands which were branched, and the xylogenesis pattern was the same regardless of the cytokinin employed. The xylogenesis pattern produced by 2,4‐D‐cytokinin varied with the 2,4‐D concentration, and was independent of the cytokinin employed. Small numbers of tracheids were observed in explants cultured under xylogenic conditions and treated with caffeine (1000 mg/liter) for the inhibition of cytokinesis.

INVESTIGATIONS ON THE ROOT FORMATION IN THE TISSUES OF HELIANTHUS TUBEROSUS CULTURED IN VITRO

Excised rhizome fragments of Helianthus tuberosus ‘Violet de Rennes’ produce roots in vitro; the sequence of morphogenetic phenomena is as follows: proliferation, differentiation of phloem and tracheids, organization of cambiums, and formation of root primordia from cambiums. This rhizogenesis is conditioned by five limiting factors: mineral salts, sugar, auxin, temperature, and light. The optimum level of each of these factors was determined and in certain cases also the threshold of action. For white light the beginning was about 1/1000 lux. These five limiting factors do not represent the same site of action. The mineral salts, especially nitrogen and calcium, have their effect when the cambiums have been organized. The sugars act before the formation of cambiums. Light operates principally upon cambium and this action is independent of photosynthesis. It was not possible to determine whether the auxin has a preferential site of action. A temperature higher than 15 C is essential to obtain the formation of cambiums but further production of roots is possible at 15 C or even less. The variations in temperature more or less around the optimum stimulate rhizogenesis. The supra‐optimal temperatures mainly have their effect before the formation of cambiums and infra‐optimal temperatures after. The effect of light can be exhibited at low temperature. Gibberellic acid in association with auxin favors root formation in the dark whereas in light it exhibits inhibiting properties. Rhizogenetic potentialities of tissues are reduced when these are cultured in the dark in presence of auxin. On the other hand, if auxin is applied after the isolation of explants, rhizogenesis is stimulated and the tissues are able to produce roots even in the dark. The action of light and of auxin can be dissociated; when the explants having absorbed a small quantity of auxin are transferred to a medium without auxin for 60 days in the dark, they conserve the rhizogenetic potential which is expressible on transferring them to light. Finally, grafting experiments suggested that light could induce the formation of a rhizogenetic factor in the tissues which is transmittable from cell to cell. These experiments have definitely established that the induction of root formation is governed by several factors which have complex interactions.

THE LIGNIN OF POPULUS NIGRA L. CV. ‘ITALICA’

Anatomical investigation showed that callus cultures of Populus nigra L. cv. ‘Italica’ contain lignified parenchyma cells and that large amounts of lignin are localized in intercellular spaces. A tissue-culture strain on a medium containing IAA also showed differentiation of tracheids with lignified walls. In the plant, lignin was found in the primary xylem, secondary xylem, phloem, and periderm. The lignin from primary xylem gave a negative Mäule reaction. Lignins from callus cultures and from the plant were compared and quantitatively determined. A method was developed to calculate the lignin content of tissues from the lignothioglycolic acid yield and the absorption at 250 nm in the so-called difference spectrum. Oxidation with nitrobenzene yielded syringaldehyde (S) and vanillin (V) from tissue cultures, young and old secondary xylem, phloem, and periderm; primary xylem gave vanillin but no syringaldehyde. For tissue cultures, young secondary xylem, phloem, and periderm, the S/V ratio is lower than for differentiated wood. For 2,4-D cultures the S/V ratio was lower than for a strain cultured on an IAA medium. The aldehyde yield, calculated as a percentage of the lignin, is appreciably higher for secondary xylem (42%) than for phloem, periderm, and tissue cultures (12–18%). Xylem lignin contains 9–10% esterified p-hydroxybenzoic acid. Less of this acid is found in the lignin of young secondary xylem (5.2%) and the tissue cultures (2.8–6.7%). Phloem lignin has a still lower p-hydroxybenzoic acid content (0.6%). Lignin rich in p-hydroxybenzoic acid is characterized by a very high absorption in the difference spectrum at 296 nm, strong absorption in the infrared spectrum at 1265 cm-1, and an extra absorption band at 766 cm-1. After hydrolyzation, this lignin lacks the 766 band in the IR spectrum and shows much less absorption at 1265 cm–1. Alkali lignin was prepared by a 16-hour extraction with 0.5 N NaOH at 70 °C. Complete extraction of the lignin from the tissue samples was rarely obtained. Lignin preparations deriving from the various tissue regions of the plant and from the callus cultures show mutual differences. The lignin of the callus cultures shows the greatest resemblance to the lignin of young secondary xylem but is much more resistant to oxidation. In this respect it shows a closer relationship to the lignin from the phloem and primary xylem of the tree. The terms “angiosperm lignin” and “hardwood lignin” applied to a lignin with a high syringaldehyde content are very misleading, because not all the lignified tissues of dicotyledonous trees contain this type of lignin.

Tracheid differentiation in tobacco pith cultures

Sterile pith cultures of Nicotiana tabacum have been induced to form localized regions of differentiating tracheids. These localized regions have been examined by phase, fluorescence, and electron microscopy, and polarization optics. Fixation for electron microscopy was with glutaraldehyde-osmium. The differentiating tracheids develop characteristic thick cell walls which are eventually lignified. The lignifications appear to be uniform throughout the secondary wall and little or no lignin appears to be deposited in the primary walls or intercellular layer. At all stages of secondary wall deposition, the peripheral cytoplasm contains a system of microtubules which form a pattern similar to that of the developing thickenings. Within this system the microtubules are oriented, the direction of orientation mirroring that of the fibrils in the most recently deposited parts of the wall. The observations support the view that the microtubules are somehow involved in microfibril orientation. The microtubules appear to be attached to the plasma membrane which has a triple layered structure. The two electron dense layers of the plasma membrane have a particulate structure. In the differentiating tracheids at regions where secondary wall thickening has not yet been deposited numerous invaginations of the plasma membrane are observed which contain loosely organized fibrillar material. It is suggested that these are areas of localized activity of the plasma membrane and that the enzymes concerned with the final organization of the cellulose microfibrils are situated at the surface of the plasma membrane. Dictyosomes in the differentiation cells give rise to vesicles which contain fibrous material and the contents are incorporated into the cell wall. Numerous profiles characteristic of plasmodesmata are evident in sections of the secondary thickenings.

The nature of reaction wood. VIII. The structure and differentiation of compression wood

The cell wall organization of tracheids of natural and chemically induced compression wood of Pinus radiata and Actinostrobus pyramidalis has been shown to be the same, and is similar to that established in previous studies of natural compression wood. In the secondary wall only two layers were present. In the second of these there was a well-developed system of helical cavities, separating ribs of cellulose. The ribs of cellulose were parallel to the direction of microfibril orientation; were complex in form; and the cellulose lamellae lay parallel with the wall surface. A well-developed wart structure was present. During the differentiation of compression wood tracheids, the intercellular spaces were formed during the phase of surface enlargement of the differentiating tracheids. At an early stage the intercellular spaces appeared to contain cytoplasmic ground substance. During the development of the layer S1 the cytoplasmic organization was similar to that of normal tracheids, the cells containing a large vacuole with a well-developed tonoplast and plasmalemma. During the development of the layer S2 the cytoplasm contained numerous small vesicles with no large vacuoles, and in many instances the plasmalemma was absent. At the conclusion of the differentiation of the cell the plasmalemma was again present and penetrated the helical cavities of the wall. Compression wood induced by 3-indoleacetic acid (IAA) alone, gibberellic acid (GA) alone, or IAA and GA in combination was identical with that formed under natural conditions. The localized lateral application of IAA to vertical stems caused conspicuous bending of the stem as well as compression wood formation.

The mechanism of surface growth involved in the differentiation of fibres and tracheids

An electron microscopic investigation has been made of the differentiating xylem elements of Pinus radiata, Eucalyptus elaeophora, and Ulmus sp. In the tips of fibres and tracheids there is a tendency for the microfibrils of cellulose to be oriented in the direction of growth. It is considered that this orientation can be disturbed by subsequent dimensional changes in the cell. The thin areas of the differentiating cells which are involved in the so-called "mosaic growth" have been compared with the regions of the cell wall penetrated by plasmodesmata in the storage parenchyma of potato tubers. The suggestion is made that the thin areas are regions of the cell wall penetrated by plasmodesmata, or are developing primary pit fields. The implications of this concept, with respect to intercellular readjustment and to the differences between fibres and tracheids in extension growth, are discussed.