THE TIME COURSE OF SIEVE TUBE AND VESSEL REGENERATION AND THEIR RELATION TO PHLOEM ANASTOMOSES IN MATURE INTERNODES OF COLEUS

Quantitative counts of regenerative sieve tubes and vessels were made in a large number of samples of mature internode #5 of C. blumei, with concomitant study of the fine details of vascular regeneration and the occurrence of the normally developing phloem anastomoses. Such anastomoses were found in many of the plants, but their average number in the small regenerating area was low (viz., 0.9 ± 0.2). With the phloem anastomoses excluded from the counts, the time course of regeneration was clear cut—no strands completed their regeneration around the wound until three days after wounding. More regenerative sieve tubes completed their differentiation under all conditions than did regenerative vessels. The number of sieve tubes and vessels regenerated by four days was closely related to the number of preexisting bundles of that type of vascular cell that had been severed by the transverse wound. The ratio of bundles severed by the wound in the phloem to those in the xylem was 2.14, and the ratio of the regenerative sieve tubes to the regenerative vessels was 2.24. For both tracheary and sieve tube cells the initial regeneration was strongly polar (mostly above the wound), as expected from earlier IAA transport data. The path of tracheary regeneration was obviously related to that of the sieve tubes on the other side of the cambium.     

Vascular Regeneration and Long Distance Transport of Indole-3-acetic Acid in Coleus Stems

Excision of all leaves and buds of Coleus blumei Benth. plants reduced xylem cell and sieve tube regeneration a highly significant amount around a wound in internode number 5 when compared with regeneration in intact (wounded) plants. Application of indoleacetic acid (IAA-¹⁴C) to the cut surface of internode number 2 restores regenerative activity around the wound in internode number 5. Radioactivity applied as IAA-¹⁴C reaches the wound area when applied at the cut surface of inter-node number 2 showing a logarithmic decrease with distance from the point of application. Chromatography showed that radioactivity was located close to the R_F of IAA as well as near the solvent front.     

Effects of auxin on the spatial distribution of cell division and xylogenesis in lettuce pith explants

Summary Cylinders of pith parenchyma were tissue-cultured with their opposite ends on media which differed only in content of the morphogens auxin (IAA), sucrose, or zeatin. A range of concentrations of each of these morphogens applied at one end (none at the other end) resulted in distribution patterns of cell division and xylogenesis that were attributable to interaction between inductive levels and morphogen mobility. Auxin was crucial for tracheary patterns: large tracheary elements formed by direct differentiation of pith cells near the auxin source, smaller but still roughly isodiametric tracheary elements formed after cell division, and tracheary strands developed where, presumably, auxin transport had become polarized and then canalized. Xylogenesis was confined to regions within millimeters of the auxin source, and [¹⁴C]IAA studies showed a steep logarithmic concentration gradient along the cylinder. Patterns of tracheary strands and rings revealed that the pith explants retained some polarity from the stem from which they had been excised. However, the direction of flow of applied auxin was more effective than original polarity in controlling the orientation of tracheary strands and their constituent tracheary elements. It seems that, in tissues with little or no polarity, diffusive flow of auxin gradually induces polar flow in the same direction, together with an associated bioelectric current, and that this orients the cortical microtubules that in turn determine the orientations of cell elongation and of the secondary wall banding in tracheary elements.Abbreviations IAA indoleacetic acid - NAA naphthaleneacetic acid - TIBA triiodobenzoic acid Dedicated to the memory of Professor John G. Torrey     

Transport of indoleacetic acid in intact roots of Phaseolus coccineus

Summary Indoleacetic acid (IAA)-5-³H (2×10^-9) was applied to intact roots of Phaseolus coccineus seedlings at the apex or 2 cm above the apex, and the movement of IAA-³H and its metabolites traced by sectioning and chromatography. Basipetal movement of label occurred for 2 cm or less, declining exponentially, and the amount increased with time. Acropetal transport from above the apex showed quantitatively less movement of radioactivity. After a 6h treatment period a decline of label occurred in the first 0.5cm, below which there was a long distance movement of small amounts of label, mainly in IAA, towards the apex where the label concentrated by a factor of approximately 2. Short-distance basipetal movement consisted of about equal amounts of IAA and metabolites, and only metabolites were found in areas more basipetal than 2cm. Label from solutions of sucrose-¹⁴C and ³H₂O followed the same general pattern of movement as label from IAA-³H, except that acropetal movement of water showed a steady decrease in the amount of label as the distance from the area of application increased. The short distance basipetal transport of label with the breakdown of IAA-³H indicates that the extent of basipetal movement was limited by catabolic processes. The acropetal pattern of IAA-³H movement with the concentration of the transported material close to the apex, is possibly the result of transport in the phloem.     

POLARITY OF INDUCTION AND PATTERN OF PRIMARY PHLOEM FIBER DIFFERENTIATION IN COLEUS

The differentiation of primary phloem fibers was studied in Coleus blumei on a quantitative basis. The pattern of fiber differentiation in intact, untreated plants was found to be in the acropetal direction (from a mature internode to a young one). The youngest internodes to differentiate primary phloem fibers were those with cambial activity. In plants grown in the winter, fibers started to differentiate in internodes closer to leaf #2 than in spring-grown plants. A wound changes the pattern of fiber differentiation surrounding it. A wound in which the tissues above and below it were separated with parafilm, prevented fiber differentiation in the tissues directly below the wound, and caused more fiber differentiation in the tissues above and lateral to it. Under wounds with no parafilm separation, few or many fibers differentiated depending on the angle of the wound. The number of fibers under diagonal wounds was five to nine times more than under a horizontal wound. By excision experiments it was found that mature leaves were the source of induction of fiber differentiation. Leaves that produced induction caused fiber differentiation in the internode below them but did not cause fiber differentiation in the internode above. The induction, which can flow through a wound and cause fiber differentiation in at least two internodes below the source, is a polar induction in the basipetal direction (i.e., in the direction from the leaves to the root). Phloem fibers differentiated only in the vascular strands and not from the parenchyma cells between the strands. Therefore, they follow the new regenerative sieve and vessel elements in the pre-existing vascular strands, but do not follow them in their regeneration between the longitudinal strands.     

Patterns of Tracheary Differentiation in Lettuce Pith Explants: Positional Control and Temperature Effects

When lettuce pith explants were cultured for 14 d on a xylogenicmedium, tracheary elements differentiated in greatest numbersbetween 25 and 30 °C. Numbers were depressed at lower temperaturesby slower development and at higher temperatures by adverseprocesses. The data did not support previous suggestions ofa great stimulation of xylogenesis above 30 °C and of aspecial sensitivity to low temperatures. Tracheary elements differentiated in various spatial patterns:as clumps in the peripheral callus, as strands which extendedradially and longitudinally from some of these clumps, as individuallarge tracheids especially at the more extreme temperatures,and as short strands associated with nodules and roots thatformed at favourable temperatures. We suggest that indoleacetic acid (IAA) has various roles inthe positional control of these tracheary patterns: (1) IAAdestruction at the explant surface leads to concentration gradientsthat inhibit tracheary induction close to the surface; (2) IAAtransport from the source in the culture medium to sinks especiallyat the explant surface, coupled with autocatalytic flow facilitation,leads to canalization along pathways that become meristematicand then trachcary strands; (3) the IAA flux (and associatedproton flux) along these pathways tend to orient cortical microtubulesat right angles to the flow, by some mechanism as yet unknown,and hence to control the orientation of tracheary element elongationand secondary wall banding. These suggestions, supported bymorphometric studies of tracheary element dimensions and orientations,and by experiments with localized IAA application, lead to ageneral interpretation of the development of polarity in plants. IAA, Lactuca sativa, lettuce, pith explants, positional control, temperature effects, tracheary element differentiation     

The biosynthesis and translocation of 14C-IAA in Ricinus communis

The biosynthesis of ¹⁴C-IAA from ¹⁴C-tryptophan applied to abraded leaves of Ricinus communis and its subsequent export through the phloem were studied. Phloem sap was collected at intervals from incisions made in the stem below the IAA fed leaf. Any upward movement of label through the phloem or downward movement of phloem mobile compounds from leaves above the treated one were restricted by bark-ringing the plants.TLC and HPLC analyses of the collected sap indicate that some conversion of ¹⁴C-tryptophan to ¹⁴C-IAA had occurred. Subsequent GC-MS analysis of the HPLC purified samples of phloem sap revealed high levels of endogenous IAA transported from the fed leaf. The high ratio of unlabelled/labelled IAA in the phloem sap makes unequivocal confirmation by GC-MS of the predicted biosynthesis of ¹⁴C-IAA impossible. It is postulated that IAA is synthesised from tryptophan in mature leaves and exported to developing sink tissues with the flow of photoassimilates in the phloem.     

Polarity of IAA Effect on Sieve-Tube and Xylem Regeneration in Coleus and Tomato Stems

A technique is described for the processing of regenerated xylem and sieve tubes from the same wound area for microscopic and quantitative comparison.

Regeneration was examined in internodes of 2 developmental stages in Coleus: internode 2, elongating, characteristic of primary growth; and internode 5, non-elongating, characteristic of secondary growth.

Transport of indoleacetic acid (IAA) in excised number 5 internodes of Coleus is strictly polar, in a basipetal direction, judging by a regeneration bioassay involving both sieve tube strands and xylem cells. Similar results were obtained with tomato.

If isolated number 5 Coleus internodes are not treated with hormone, they regenerate no xylem cells and a small number of sieve tube strands. With increasing concentrations of IAA added apically, the number of regenerated sieve tube strands (and, with higher concentrations, of xylem cells) increases progressively up to 1% IAA, the highest concentration applied.

Internode 2 of Coleus regenerates fewer xylem cells or sieve tube strands than internode 5, whether on the otherwise intact plant or with a given concentration of IAA added apically. The amount of regenerated xylem increases with added apical IAA, except that the highest concentration gives no further increase. The number of xylem cells regenerated in intact plants occurs at the same interpolated IAA concentration as in number 5 internodes. No concentration of IAA tried provided replacement of intact number of sieve tube strands in internode 2.

IAA can exert a regenerative stimulus on both xylem and sieve tubes in the area immediately adjacent to the site of its application.

     

Naturally Occurring Regenerative Differentiation of Xylem Around Adventitious Roots in Luffa cylindrica Seedlings

Regeneration of xylem induced by adventitious root formationin the hypocotyl of Luffa cylindrica Roem. seedlings is described.This naturally occurring form of xylem regeneration involvesthe formation of a bypass of regenerated tracheary elementsaround a root without external severance of the vascular strands.The regeneration of xylem around an adventitious root is polarand is very similar in its developmental pattern to the well-knownxylem regeneration induced by wounding vascular strands. Adventitious root formation, Luffa cylindrica Roem, regenerated tracheary elements, vascular differentiation, xylem regeneration     

Naturally Occurring Regenerative Differentiation of Xylem Around Adventitious Roots in Luffa cylindrica Seedlings

Regeneration of xylem induced by adventitious root formationin the hypocotyl of Luff a cylindrica Roem. seedlings is described.This naturally occurring form of xylem regeneration involvesthe formation of a bypass of regenerated tracheary elementsaround a root without external severance of the vascular strands.The regeneration of xylem around an adventitious root is polarand is very similar in its developmental pattern to the well-knownxylem regeneration induced by wounding vascular strands. Adventitious root formation, Luffa cylindrica Roem, regenerated tracheary elements, vascular differentiation, xylem regeneration.     

Effects of Auxin Concentration on the Dimensions and Patterns of Tracheary Elements Differentiating in Pith Explants

The optimal concentration of IAA (0.03 mM) for tracheary elementdifferentiation in lettuce pith explants was about ten timesgreater than the optimal concentration for callus proliferation.Related to this, the mean volume per tracheary element increasedwith increasing IAA concentration, 18-fold between 0.001 mMand 0.3 mM IAA. At the highest concentrations, some pith cellsappeared to differentiate directly into tracheary elements,without cell division, resulting in especially large trachearyelements. Tracheary strands developed at intermediate concentrationsof IAA, and led to a small increase in the mean length/breadthratio of tracheary elements. For tracheary elements differentiating from stem cambial derivatives,a reassessment of previous studies indicates that increase inauxin concentration brings greater tracheary element size atconcentrations up to the 0.03 mM optimum. Above this optimum,however, further increase in auxin concentration brings progressivelysmaller tracheary elements, as the high auxin curtails enlargementof the differentiating cells. This contrasts with the pith explants,in which tracheary element size increases with IAA concentrationmost markedly above the optimum concentration. The interpretationof these relations requires an understanding of the effectsof auxin concentration on interacting quantities such as initialsize of cells, rate of enlargement, and rate of differentiation. Lactuca sativa, lettuce, IAA concentration, pith explants, tracheary element dimensions     

High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon)

A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin (6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv. Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z and 0.5 mg l⁻¹ IAA + 2 mg l⁻¹ Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l⁻¹ NAA + 2 mg l⁻¹ Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation.     

Polar Movement of Indole-3-acetic Acid-C in Roots of Lens and Phaseolus

A critical review of the few papers on IAA-¹⁴C movement in roots revealed apparent contradictions, as well as flaws in experimental design that would be apt to cause artifacts. The movement of ¹⁴C from IAA-¹⁴C was studied in sections of Lens and Phaseolus roots, using a system 20 or more times as sensitive as any previously used. To make sure that our results with roots could be compared validly with published work on petioles and stems, we used the same techniques as we had earlier used for shoot structures. The results with Lens were similar in many ways to those for shoots: net movement into receiver blocks was very strongly polar, followed a linear course for several hours, and showed a velocity of the same order of magnitude as in shoots (and, in fact, very close in absolute value to that found in Coleus stem cylinders). Also, as with shoots, all the radioactivity in receiver blocks ran to the R_F of IAA. The time-course of loss of counts from donor blocks was similar to that found in shoots. The 2 most striking differences from shoots were 1) the very low percentage of added ¹⁴C that was moved into the receivers (about one-tenth of the values for bean petioles), and 2) the fact that the polar movement was acropetal in roots, rather than basipetal as in shoots. Results with Phaseolus roots were similar to those for Lens, although an additional complication with Phaseolus roots was the indication of a transitory stage of weak basipetal polarity in the first few hours after excising the section. This stage was followed in a few hours by a stronger acropetal polarity.     

HORMONAL CONTROL OF TRANSLOCATION OF PHOTOSYNTHETICALLY ASSIMILATED 14C IN YOUNG SOYBEAN PLANTS

The normal supply of growth substances to a young soybean plant was altered by removing the plant's apical meristem and replacing this meristem with an aqueous solution of either indole-3-acetic acid (IAA), gibberellic acid (GA), or water. The length of each experiment was 1 hr. In the middle of it, ¹⁴CO₂ was administered to one of the primary leaves of the plant, and at the end distribution of ¹⁴C in various parts of the plant was determined. It was found that an addition of growth substances stimulated translocation in three different ways. Both IAA and GA increased the total amounts of sucrose-¹⁴C translocated, increased the rate of its translocation, and affected the distribution pattern of translocated sucrose throughout the plant. Experiments using IAA-¹⁴C have shown that the action of IAA is on the longitudinal translocation in the stem and not on the transfer of photosynthate from the mesophyll to the conducting tissues of the leaf.     

Selection of hydroxyproline-resistant cell lines from Solanum Tuberosum L. Callus. II. Plant regeneration and frost tolerance of regenerated plants

The calluses of two hydroxyproline-resistant lines (D20-1 and D30-1) of Solanum tuberosum L. were transferred to a solidified MS medium containing 1.0 mg/I IAA, 2.0 mg/l zeatin, 40.0 mg/l adenine sulphate, 1 g/l casein hydrolysate, 20 g/l sucrose and 10 g/l agar for plant regeneration. The shoot regeneration was only achieved from the callus of line D20–1. Regenerated shoots exhibited morphological variability. The degrees of frost tolerance were higher in the leaves of the regenerated plants compared with the leaves of the non-selected control plants, but lower than that of the callus from which they were regenerated.     

IAA Amino Acid Conjugates Induce Differentiation of Tracheary Strands in Lettuce Pith Explants

Each of four amino acid conjugates of IAA was able to replacethe IAA requirement for xylogenesis in lettuce pith explants,when supplied at concentrations ten to 100 times those optimalfor IAA. Tracheary development induced by these conjugates tendedto be slightly slower and less in amount than with IAA, andthe tracheary strands shorter and less regular. Responses differedsomewhat among the four conjugates: IAA-D, L-aspartate gavedevelopment most like that with free IAA, and IAA-D, L-phenylalanineoften yielded the weakest tracheary development, while responsesto IAA-L-alanine and IAA-glycine were intermediate. The resultsare interpreted in terms of the ‘bound’ IAA conjugatesdiffusing into the pith explants and becoming xylogenic onlyon hydrolysis to ‘free’ IAA. As tracheary strandformation is believed to result from IAA fluxes, it seems thatthe free IAA also moved through the discs, presumably towardsthe surfaces where it degrades rapidly. Tracheary strand formationin these explants can be compared with vascular strand formationin the normal shoot tip, where IAA conjugates (auxin ‘precursors’)move acropetally and are hydrolysed to free IAA especially inthe young leaf primordia, we suggest, yielding local sourcesof IAA which may contribute both to the phyllotactic spacingof primordia and, moving basipetally, to the definition of theauxin pathways that develop as procambial strands behind individualleaf primordia. Lactuca sativa, lettuce, IAA conjugates, tracheary element differentiation, pith explants, xylem strands     

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of psophocarpus tetragonolobus (L.) DC

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L⁻¹ induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L⁻¹) and IAA (0.2 mg L⁻¹) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.     

Direct shoot regeneration from mature embryo as a rapid and genotype-independent pathway in tissue culture of heterogeneous diverse sets of cumin (Cuminum cyminum L.) genotypes

Summary A rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15–22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mgl^−1 (4.3 μM) NAA in combination with 0.3 mgl^−1 (1.71 μM) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.     

Translocation of Shoot-Applied Indolylacetic Acid into the Roots of Populus tremula

Application of 10 to 100 μg indol-3-ylacetic acid to the leaves of rooted cuttings of aspen caused inhibition of root growth after three hours. Root growth recovered within 24 hours after IAA treatment. Swelling of the root tips occurred during the period of inhibition. The roots responded in the same way if IAA was applied in solution to the cut stem surface above the mature leaves. IAA-1-¹⁴C applied through a cut stem surface or to mature leaves was translocated downwards in the plants and labelled IAA could be isolated from the roots 3 to 24 hours after application. The ethanol-soluble activity decreased rapidly indicating a rapid metabolism or binding of IAA. IAA-1-¹⁴C applied to growing leaves was not translocated. From the rapid response of root growth it was concluded that IAA was translocated into the roots at a rate of about 7 cm per hour. This rate of translocation indicates that the sieve tubes are involved in the translocation. Implications of the results for the translocation of endogenous auxin into the roots are discussed.     

Evidence for Three Different Systems of Movement of Indoleacetic Acid in Intact Roots of Phaseolus coccineus

Indoleacetic acid (IAA)-5-³H (2 × 10⁻⁹M) was applied to intact roots of Phaseolus coccineus seedlings, at the apex or 2 cm above the apex, at various pHs and in the presence of Cu²⁺ and NaCl. The transport of label in the roots was then examined after 6 h by cutting the roots into 1 mm sections above and below the zone of treatment. Basipetal movement from 2 cm above the apex was unafected by pH, Cu²⁺ or NaCl. Acropetal movement from the same area decreased with increasing pH from 5.4 to 8.0, probably due to an effect of pH on the entry of IAA into the cells. pH had no effect on sucrose transport. Cu²⁺ also inhibited acropetal movement but NaCl had no effect. Basipetal movement of label from the apex was reduced by Cu²⁺ and increasing pH, but not as much as with acropetal movement, and increased by the presence of NaCl. These facts are interpreted as showing 3 different systems of IAA movement in intact roots: basipetal from 2 cm up the root in some extracellular physical system; acropetal from 2 cm up the root, and basipetal from the apex, in a metabolically dependent intracellular system, but in different tissues of the root. It is proposed that endogenous IAA not only moves into the root from the stem but is also synthesized in the root apex, and moves basipetally for a short distance to the root growing zone in a separate system from the IAA descending from the stem.