Auxin Ratio Research Articles

Control of Morphogenesis in Sorghum by 2,4-Dichlorophenoxyacetic Acid and Cytokinins

Dichlorophenoxyacetic acid caused a shortening of roots and shoots when mature seeds of Sorghum bicolor (L.) Moench cv. BTx3197 were germinated on Murashige and Skoog (MS) medium that contained 2,4-D. Shoot growth was restored with cytokinins. A callus formed at the nodal region, the further differentiation of which was determined by the ratio of 2,4-D and cytokinins in the initial culture medium. A high auxin to cytokinin ratio promoted primarily root differentiation while a high cytokinin to auxin ratio promoted multiple bud development. Isolated shoot apical meristem with the subtending node produced embryogenic callus at low cytokinin levels and green buds on high cytokinin levels when cultured in the presence of 2,4-D. It is concluded that cells potentially capable of differentiation into roots, somatic embryos or axillary buds are present in the first nodal region.

Similarities between the effects of aphid infestation and cytokinin application on dark respiration and plant growth of legumes

Cowpea (Vigna unguiculata (L.) Walp. cv. Caloona), broad bean (Vicia faba L. cv. Aquadulce), and garden pea (Pisum sativum L. cv. Victory Freezer) seedlings were infested with cowpea aphids (Aphis craccivora Koch) or pea aphids (Acyrthosiphon pisum (Harris)), both Homoptera: Aphididae, for 10 days and then infested host plant tissue was examined for foreign substances injected by the aphids. No foreign compound was detected in any of the aphid-infested plant tissues. Both aphid species were also assayed for plant growth substances, utilizing the epinastic response of tomato (Lycopersicon esculentum Mill. cv. Roma Teardrop) seedlings, and both aphid species contained plant growth substances in concentrations higher than plant physiological concentrations. Broad bean and pea seedlings were also treated with foliar and root applications of 6-benzylaminopurine to determine if there were any similarities in plant growth or respiratory responses, following aphid infestation or 6-benzylaminopurine treatment. Root respiration in 6-benzylaminopurine treated plants decreased while shoot respiration increased in a response analogous to that observed for aphid-infested tissue. However, the alternative respiratory pathway was engaged for all 6-benzylaminopurine treatments, whereas in aphid-infested plant roots and shoots it was not. Both 6-benzylaminopurine treated and aphid-infested plants displayed a loss of apical dominance. These data suggest that part of the physiological response of the plant to aphid feeding is induced by changes in the cytokinin to auxin ratio.

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. Green Comet was used in a study of the effects of exogenous hormones on the growth and differentiation of seedling organs in vitro. Four types of explants were tested: hypocotyl segments, root segments, primary leaf discs and cotyledon discs. These explants were incubated on media containing factorial combinations of BAP x IBA, BAP x NAA, KN x IBA and KN x NAA(all at 0, 0.1, 1.0 and 10.0mgl-1). Hypocotyls were the most regenerative explants; shoot production was favoured by cytokinin:auxin ratios greater than one and was decreased by IBA at 10 mg I1 when callus was produced. Shoot formation from root explants occurred either in the absence of hormones or with low concentrations; no shoot was produced when any hormone was present at lOmgl-1. In contrast, shoot production from primary leaf discs was favoured by high concentrations of both auxin and cytokinin with the combination of BAP and IBA the most effective. Shoot production from cotyledon discs was sporadic with no consistent response on any auxin/cytokinin combination. After further experiments on the optimization of hormone concentration, the following combinations were chosen as allowing reliable regeneration:0.1 mg l-1 BAP+0.lmgl-1 IBA for hypocotyl segments, 0.075 mg l-1 K.N + 0.025 mgl-1 IBA for root segments, and 5.0 mg l-1 BAP+5.0 mg l-1 IBA for leaf discs.

Manipulation of the morphogenetic pathways of tobacco explants by oligosaccharins

Acquiring the ability to regulate morphogenesis in plants has long been an elusive goal. Previous research has established that ‘thin cell-layer’ explants from the surface of the floral branches of tobacco can be induced to form either callus, vegetative buds, flowers or roots by adjusting the pH and the ratio of auxin to cytokinin in the culture medium1–5. We now report that oligosaccharins (oligosaccharides with regulatory activity) derived from plant cell walls can, at concentrations of ∼10−8 or 10−9 M, induce tobacco expiants to form vegetative buds instead of flowers or callus, or flowers instead of vegetative buds. Other oligosaccharins can induce the expiants to form roots instead of vegetative buds. This ability of oligosaccharins to control morphogenesis suggests that these fragments are in situ regulators of morphogenesis.

High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4‐day‐old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4‐D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).

The stimulatory effect of liquid induction medium on shoot proliferation of Ruscus hypophyllum L.

Pre-culture of shoot tip and inflorescence explants of Ruscus hypophyllum (popular names are “butcher's broom” or “mouse thorn”) in shake liquid culture increased both axillary and adventitious shoot proliferation 6–10 fold. Explants pre-cultured in agitated Linsmaier and Skoog liquid medium containing 1 mg 1 −1 each of 2,4-dichloroacetic acid and 6-benzylamino purine had to be sub-cultured after 15 days to a solid medium with a higher (5:1) cytokinin: auxin ratio for shoot development. Explants remaining in liquid medium increased in size and produced only few shoots. The initial agitation in liquid medium provided the explants with optimum contact with the medium and the subsequent release from apical dominance after transfer to stationary culture. In addition, the agitation in liquid medium induced adventitious shoot proliferation from the epidermal tissue of the phylloclades.

Simultaneous inhibition and induction of compression wood formation by morphactin in artificially inclined stems of Japanese larch (Larix leptolepis Gordon)

Morphactin IT3456, applied in a 2–3 cm band around the middle portion of 2- or 3-year-old internodes of artificially inclined 5-year-old Japanese larches, induced compression wood formation on both the upper and the lower sides of the inclined stem above the treated region, while it inhibited compression wood formation below this region. These results seem to suggest that a high concentration ratio of endogenous auxin to sugar (auxin/sugar) in the differentiating xylem tissue is necessary and sufficient for compression wood formation, and that compression wood formation under natural conditions requires polar transport of auxin which supplies and maintains high concentration of auxin along the undeside of the stem.

Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.)

Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. ;Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10(-5) and 10(-6) molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10(-5) and 10(-6) molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin alpha (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system.

Root formation in pea cuttings: Effects of combined application of auxin and cytokinin at different developmental stages

Cuttings of pea cv. Alaska and ov. Kelwo were both decapitated and disbudded at different time intervals after cutting. Auxin and cytokinin combined in different ratios were applied to the upper part of the decapitated and disbudded cuttings. The effects of different ratios of auxin and cytokinin were not the same when applied at different developmental stages of the root initiation phase. The results seem to demonstrate an interaction between auxin and cytokinin at different ratios throughout the root initiation phase. The effects of combined application of auxin and cytokinin suggest that different stages of the root initiation phase require different levels of auxin and cytokinin. A higher level of auxin and either lower or equal level of cytokinin may be needed only in the early stages. During the subsequent stages a lower level of auxin in combination with a higher level of cytokinin seems to be more conducive.

Caryological stability of Datura innoxia calli analysed by cytophotometry for 22 hormonal combinations

Cell lines from microspore-derived embryoids of Datura innoxia were maintained in vitro under 22 different hormonal combinations and periodically submitted to cytophotometric measurements in order to check their caryological stability. Among the combinations tested, some, with a high cytokinin : auxin ratio, showed a tendency to select for the proliferation of preferentially low DNA content nuclei. These results, corroborated by a proper statistical analysis of the data, could be the consequence of two concomitant phenomena: the selective advantage of haploid nuclei due to their shorter mitotic cycle, together with an increased stimulation to mitotic division provoked by a higher cytokinin supplement. Our data, in accordance with those obtained in two other laboratories, seem to indicate that Datura innoxia cell lines can maintain a low DNA content level in their nuclei and easily reach a satisfactory caryological stability in vitro.

In vitro propagation of watermelon

A 3-stage, in vitro propagation system for diploid watermelon ( Citrullus lanatus (Thumb.) Matsum. and Nakai) has been developed which is also applicable to the economic production of triploid (seedless) watermelon transplants. Stage I involves the stimulation of axillary-bud development from excised seedling shoot tips (1–3 mm) by a high cytokinin (K)/low auxin (IAA) ratio (4.6 μmol/l K 0.28 μmol/l IAA) on a modified Linsmaier and Skoog medium. An average of 4.5 axillary shoots of sufficient size to subculture (> 15 mm) were obtained from each explant in 5 weeks. These shoots were induced to root during 3 weeks subculture on Stage II medium containing 11.5 μmol/l IAA. Well-rooted plantlets were successfully transplanted to a 1 : 1 peat : sand (v/v) substrate and gradually “hardened-off” to greenhouse conditions (Stage III) for 3 weeks, after which they could be transferred to field conditions. Alternatively, axillary shoots from Stage I could be returned to fresh media with 9.3 μmol/l K and 0.28 μmol/l IAA, where an average of 10.3 axillary shoots could be obtained after 5 weeks. Cost estimates for producing 10 000 finished transplants per week project an approximate cost of 16 dollar cents per transplant.

Bud induction on isolated needles of Norway spruce ( Picea abies L. Karst.) grown in vitro

Isolated needles of Picea abies L., Karst. were induced to form adventitious bud primordia when cultured on media containing a relatively high cytokinin: auxin ratio. In order to receive a high frequency of needles forming bud primordia it was necessary to collect the needles from buds just after flushing. Short needles (1–3 mm) more readily formed bud primordia than long needles (3–5 mm). Development of primordia into buds occurred when needles bearing primordia were transferred to media either lacking growth substances or with a reduced level. A higher yield of buds was obtained if the needles were incubated at lower temperature (10–16°C) for the first days after isolation and then transferred to 20°C. Buds with 3–5 mm-long needles were excised from the initial tissue and planted individually on culture media.

The position of regenerating cambia: auxin/sucrose ratio and the gradient induction hypothesis.

To account for the positions in which vascular cambia regenerate in wound callus, a gradient induction hypothesis was proposed in 1961 in terms of gradients in 'some factor as yet unknown'. It now seems likely that the gradient is based on morphogen diffusion between source and sink on opposite sides of existing cambia, with morphogen diffusing into the adjoining wound callus. It is specifically proposed that there are two morphogens, auxin diffusing centrifugally and sucrose diffusing centripetally. The cambium then regenerates along a path where the ratio of auxin to sucrose concentration is similar to that at the original cambium, and its orientation (as regards xylem and phloem formation) is determined by the direction of the gradient in this ratio. These proposals are supported by published evidence on auxin and sucrose concentration gradients across the cambium, and on their sources, movements, and known effects on vascular differentiation. Simulations of the proposed positional control system predict patterns of cambial regeneration and orientation corresponding to those observed in four different types of wound and graft.

The influence of gibberellic acid and abscisic acid on cell and tissue differentiation of bean callus.

Bean callus was induced to form roots (tissue differentiation) and vascular nodules (cell differentiation) by lowering the ratio of auxin to cytokinin in the growth medium. Both types of differentiation were inhibited by the addition of abscisic acid (at concentrations greater than I muM) to induction medium. Initiation of differentiation was inhibited, but its subsequent development was not, and the inhibition was not affected by the addition of gibberellic acid. Addition of gibberellic acid (GA) alone to induction medium stimulated tissue differentiation, although cell differentiation was unaffected (30 muM GA) or inhibited (45 muM GA) and its onset was delayed at both concentrations. Root initiation was also stimulated by gibberellic acid (0.I-45 muM) at an auxin-to-kinin ratio 10 times that normally optimal for cell differentiation. The phenylalanine ammonia lyase (PAL) activity of the calluses was closely correlated with the amount of cell differentiation which had occurred, and measurement of this confirmed that gibberellic acid delayed the initiation of cell differentiation. The increase and subsequent decline of PAL and betaI leads to 3 glucan synthetase activities, normally induced by transfer to induction medium, was abolished by abscisic acid. Addition of gibberellic acid did not affect the betaI leads to 3 glucan synthetase activity.

Redistribution of endogenous gibberellins in geotropically stimulated roots.

THE difference in growth rate of the two sides of geotropically reacting plant organs is generally explained by the well documented unequal auxin concentrations in the two halves. The auxin ratio in horizontally placed stems, coleoptiles, and roots usually attains a value of about 2:1 in favour of the lower half.

Vegetative propagation of Alstroemeria in vitro

Young actively growing tissue explants from Alstroemeria inflorescence stem taken at a distance of 1–2 mm below the apex are capable of regenerating buds and roots from which small plantlets can be established. Root and bud regeneration was directly from the stem tissue and not from the callus. White's medium supported growth and regeneration as well as Murashige & Skoog's medium. A higher ratio of auxin to cytokinin resulted in root regeneration, while the reversed ratio promoted bud differentiation. Plantlets were obtained upon bud subculture on a low sucrose medium supplemented with indoleacetic acid but without kinetin.

Differences in ploidy and degree of intercellular contact in differentiating and non-differentiating sycamore calluses.

ABSTRACT Comparisons have been made between a sycamore callus (S4) isolated in 1970 and one first isolated in 1958 (S2). Incubation of S4 on media containing different concentrations of auxin and kinetin showed that the ratio of the hormones was the factor which controlled the growth pattern within the tissue. High ratios of auxin: cytokinin favoured a white friable tissue, whereas a compact callus with a much harder texture was formed at low ratios. Roots were differentiated at intermediate values. Callus (S2) has not been induced to undergo cellular differentiation. No soluble factors which were capable of stimulating differentiation in S2 were produced during the differentiation of S4, and S2 did not affect the ability of S4 to form roots. Divisions seen in suspension cultures of S4 callus were mainly diploid; the cells of S2 were predominantly tetrapioid. When S2 suspensions were grown in the cytokinin-free medium used for S4 a considerable number of diploid cells were seen. A number of S2 clones have been isolated and all these developed chloroplasts on exposure to continuous light and, when grown on the medium used to induce differentiation in S4, five of them showed a growth pattern in which the cells were more closely packed than is usually the case in S2 callus. A greater degree of cell contact has also been induced by including polyethylene glycol in the medium and so restricting the availability of water to the callus. Under these conditions the cells must have a greater capacity for interaction with one another. The effect of tissue texture on the extent of cell contact and adhesion between the cells is discussed in relation to the composition of the plant cell wall and ideas on the totipotency of plant tissue cultures.

Geotropic Responses in Helianthus and their Dependence on the Auxin Ratio. – With a Refined Mathematical Description of the Course of Geotropic Movements

AbstractThe paper describes a refinement of a previously published theory for geotropic movements of Helianthus annuus hypocotyls.The introduction of a logarithmic relation between growth rate and auxin concentration means an extension compared with the previously published approach. It is theoretically and experimentally shown that the refined theory can successfully be linked with reported measurements on lateral auxin transport in geotropically stimulated plant stems.The non‐linear character of the theory is discussed in connexion with theoretical descriptions on circumnutations of the hypocotyls. The refined theory is shown to predict the amplitude of the circumnutations reasonably well.

In Vitro Culture of the Monocot Narcissus triandrus L. cv. Thalia

Sterile culture of excised daffodil tissue, Narcissvs triandrgs L. cz. Thalia, was attempted on 12 media. Callus tissue and bulbets formed by differentiation of callus tissue proliferated only on media of high auxin levels. Evidence of alkaloid production by these cultures was obtained. Callus tissues growing on sterile media have provided experimental materials for many fundamental and applied investigations of higher plants. Somatic tissue from a wide variety of dicotyledonous plants have been grown iv vitro as callus, but most attempts to culture comparable tissues from Inonocotyledonous plants have been unsuccessful. With the recent reports of in ?vitro culture of somatic wheat callus tissue (1 ) and of sugar cane cells (2) ¢nly a few successful reports of previously cultured monocotyledonous tissues other than endosperm are available. The present communication deocribe3 a n.etllod for c)btaining callus cultures of daffodil tissue and preliminary investigations of its metabolic capabilities. Commercial (3) bulbs of Nczrciss triandAs L. cv. Thalia were vernalized fcor six weeks at 4°C. The bulbs were peeled of excess scales, surface sterilized with saturated calcium hypochlorite solution, rinsed three times with sterile distilled water, cut into pieces of approximately one gram weight and implanted on the selected medium. Twelve modiScations of the medium described by Murashige and Skoog (At) were tested. The medium selected for maintenance of the cultures was Murashige and Skoog's basal medium with 10 mg per liter of naphthalene acetic acid, 1 mg per liter of kinetin and 1 mg per liter of gibberellic acid. Variations consisted of changing the ratios of auxin (both naphthalene acetic acid and indole acetic acid) to kinetin and the addition of gibberellic acid and casein hydrolysate (both enzymatic and acid hydrolyzed). The cultures were grown at 27° in the dark in 125 Erlenmeyer flasks containing approximately 50 ml of agar medium. These were initially observed for growth and subcultured to fresh media when two to three fold growth had occurred. A comparison of the times Transactions of the Kansas Academy of Science, Vol. 74, No. 1, 1971. Published January 7, 1972. 1 This srork was supported in part by funds provided to the University of Kansas by the National Institutes of Health Biomedical Sciences Support Grant.

Induced morphogenesis in tissue cultures of Solanum xanthocarpum

Differentiation of shoots and roots or unorganized proliferation could be induced in tissue cultures of Solanum xanthocarpum when callus tissues habituated to a medium containing 2.4-D and meso-inositol were transferred to an auxinfree medium or to a medium in which the ratio of auxin and meso-inositol had been altered.