Potato transformation by T-DNA Cytokinin synthesis gene

Potato plants were transformed by the A.tumefaciens vector which integrates into the plant genome two foreign genes only: pTi C58 T-DNA gene 4 (ipt) responsible for elevated synthesis of cytokinins and kanamycin resistance gene. Three teratoma clones studied showed approximately 3 times, 6 times and 9 times increased levels of zeatin riboside and zeatin in comparison with untransformed controls and slight increase of the IAA level. Shoots formed roots in agar cultures sporadically, if the increase of zeatin riboside and zeatin levels was not higher than 6 times the control level. The chlorophyll content and net photosynthetic rate decreased with increasing levels of zeatin and zeatin riboside.     

Expression of a cytokinin synthesis gene from Agrobacterium tumefaciens T-DNA in basket willow (Salix viminalis)

Willow cells transformed with an ipt gene from Agrobacterium tumefaciens grow in tissue culture as undifferentiated callus without shoot induction. We show that the transformed calluses contained high levels of the cytokinins 9-β- d -ribofuranosyl zeatin and its monophosphate, demonstrating the presence of a functional isopentenyl transferase enzyme. The ipt gene was transcribed at different levels in different transformed callus lines. The absence of shoot differntiation is apparently not due to a lack of zeatin-type cytokinins in the transformed callus.     

Analysis of Cytokinin Metabolism in ipt Transgenic Tobacco by Liquid Chromatography-Tandem Mass Spectrometry

The endogenous levels of the major, naturally occurring cytokinins in Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit promoter-isopentenyl transferase gene (Pssu-ipt)-transformed tobacco (Nicotiana tabacum L.) callus were quantified using electrospray-liquid chromatography-tandem mass spectrometry during a 6-week subcultivation period. An ipt gene was expressed under control of a tetracycline-inducible promoter for a more detailed study of cytokinin accumulation and metabolism. Activation of the ipt in both expression systems resulted in the production of mainly zeatin-type cytokinins. No accumulation of isopentenyladenine or isopentenyladenosine was observed. In Pssu-ipt-transformed calli, as well as in the tetracycline-inducible ipt leaves, metabolic inactivation occurred through O-glucoside conjugation. No significant elevation of cytokinin N-glucosides levels was observed. Side-chain reduction to dihydrozeatin-type cytokinins was observed in both systems. The levels of the endogenous cytokinins varied in time and were subject to homeostatic regulatory mechanisms. Feeding experiments of ipt transgenic callus with [3H]isopentenyladenine and [3H]isopentenyladenosine mainly led to labeled adenine-like compounds, which are degradation products from cytokininoxidase activity. Incorporation of radioactivity in zeatin riboside was observed, although to a much lesser extent.     

Role for Cytokinins in Somatic Embryogenesis of Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)?

The effects of cytokinins on somatic embryogenesis in pepper were studied using a Murashige and Skoog-based medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid and 10% sucrose. Compared to the medium without cytokinins, N⁶-(Δ²-isopentenyl)-adenine and kinetin had no significant effect, whereas benzylaminopurine and zeatin significantly reduced somatic embryogenesis. Coconut water (10%) either had no significant effect or it reduced embryogenesis as well. Induction of somatic embryogenesis was also dependent on genotype. Appearance of somatic embryos continued after removal of the embryos that developed first, especially on cultivars with a lower initial induction rate. Although somatic embryos germinated, the apical meristem did not elongate.     

Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes

Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins. All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross. Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type. Unexpectedly, hormone levels in the cross were very similar to wild-type levels. Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced. The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs. As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Cowpea embryo rescue. 1. Influence of culture media composition on plant recovery from isolated immature embryos

Factors responsible for successful rescue of immature embryos of cowpea [Vigna unguiculata (L.) Walp.] and V. vexillata (L.) and for in vitro embryo development were studied. A new basal medium for embryo development in vitro was formulated on the basis of the mineral composition of embryos. Sucrose, fructose and glucose were compared as carbohydrate sources. The highest frequency of embryos developing into plants was obtained with sucrose. Adding casein hydrolysate to the medium increased plant recovery by 30%. Among the plant growth factors used, cytokinins, zeatin, 6-benzylaminopurine and kinetin were the most effective in promoting embryo maturation and development. A method that can routinely ensure high plant recovery from cultured immature cowpea embryos is proposed. Received: 4 June 1996 / Revision received: 28 October 1996 / Accepted: 22 November 1996     

The role of sucrose and different cytokinins in the in vitro floral morphogenesis of rose (hybrid tea) cv. “First Prize”

Shoots of rose (hybrid tea) cv. “First Prize” were induced to flower in vitro on Murashige and Skoog (MS) medium containing various sucrose concentrations (15, 30 or 45 g l⁻¹) and different phytohormone combinations of different cytokinins [N⁶-benzyladenine (BA); thidiazuron (TDZ) and zeatin] with α-naphthaleneacetic acid (NAA). Results indicate that sucrose is the key factor in floral morphogenesis while cytokinin increases the flowering percentage and helps the normal development of floral buds. From the three cytokinins that were used, BA and zeatin were considered to be more suitable as inductive flowering agents than TDZ. Reduced inorganic and organic salt concentration in MS media had a positive effect on in vitro flowering. The morphology of shoots bearing floral buds varied with different cytokinin treatments. The highest percentage (45%) of flowering was obtained on MS medium supplemented with 3.0 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA and 30 g l⁻¹ sucrose.     

Efficient in vitro plant regeneration from immature zygotic embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and Sorghum bicolor (L.) Moench

We report here an in vitro culture system that provides reliable, highly efficient regeneration from immature embryos of pearl millet [Pennisetum glaucum (L.) R. Br.] and sorghum [Sorghum bicolor (L.) Moench]. Immature embryos were isolated 10-20 days after pollination and cultured on various L3 media. The influence of different parameters during the callus induction phase was examined with respect to the regeneration rate: (1) the concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and various cytokinins; (2) the addition of AgNO₃; (3) the use of maltose or sucrose as a carbon source. Modifications in the phytohormones alone resulted in the regeneration of fertile sorghum plants at high efficiency. Significant increases in the regeneration rates of pearl millet genotypes were achieved by the combination of sucrose as a carbon source and silver nitrate as a potential ethylene inhibitor.     

An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.     

Changes in cytokinin activities before,during and after floral initiation in Polianthes tuberosa

The contents of endogenous cytokinin in tuberose corms (Polianthes tuberosa) at vegetative, early floral initiation, and flower development stages were investigated. We also determined the influence of exogenous cytokinin treatment on the corm apex at three different growth stages in relation to floral initiation and development in tuberose. The exogenous cytokinin effectively induced floral initiation and development, especially at the early floral initiation and flower development stages. Endogenous cytokinins were higher in early floral initiation and development stages in comparison to the vegetative stage. During floral initiation stage, the zeatin and dihydrozeatin increased significantly, while the cytokinins, zeatin riboside, dihydrozeatin riboside, ⁶N-(δ²-isopentenyl) adenine, and ⁶N-(δ²-isopentenyl) adenine riboside at consistently low levels. The increase of cytokinin levels in tuberose corms during floral induction suggests a role for cytokinins in tuberose apex evocation. Moreover, these results indicate that cytokinins seem to promote the development of flower buds rather than inducing flowering in tuberose.     

Changes in cytokinin levels of Phalaenopsis leaves at high temperature

The effect of high temperatures on cytokinin levels in Phalaenopsis hybrida leaves was investigated. Endogenous cytokinins were identified and quantified in Phalaenopsis leaves grown under high temperature conditions (30/25 °C day/night) using high performance liquid chromatography, bioassay and gas chromatography-selected ion monitoring-mass spectrometry. After 5 and 20 d of low temperature (25/20 °C day/night), zeatin, zeatin riboside and dihydrozeatin levels in the leaves were higher than that in leaves subjected to high temperature treatments. When Phalaenopsis leaves were exposed to low temperatures, about 76 % of the free cytokinins detected were of the zeatin-type. Glucoside cytokinins in the leaves increased significantly 5 d following high temperatures, and the rate of increase in glucoside cytokinins corresponded to the duration of high temperatures. At the same time, zeatin riboside and dihydrozeatin declined significantly following high temperature application. A significant accumulation of glucoside cytokinins, zeatin-9-glucoside, zeatin-O-glucoside, zeatin riboside-O-glucoside, and dihydrozeatin-O-glucoside was observed 20 d following high temperatures. These results suggest that high temperatures lead to an accumulation of glucoside cytokinins and a reduction of free base and riboside cytokinins.     

A comparative study of different cytokinins on the formation of Rhododendron forrestii Balf. f. ex diels. axillary shoots in vitro

The effect of different concentrations and activities of cytokinins on the morphogenesis of regenerated Rhododendron forrestii Balf. f. ex Diels. shoots taken from nodal segments were tested. We evaluated zeatin, zeatin riboside, izopentyladenine, izopentyladenine riboside, kinetin, kinetin riboside, benzylaminopurine, benzylaminopurine riboside. The experimental results were evaluated by mathematical methods and regression analysis describing the effect of isoprenic and aromatic type of cytokinins. On the basis of this modelling, maximum axillary shoot production was attained with medium supplemented with 2.0 mg·l⁻¹ izopentyladenine riboside, 2.0 mg·l⁻¹ benzylaminopurine and 20 g·l⁻¹ sucrose. Minimal axillary shoots were produced with kinetin and kinetin riboside.     

Hybrid Weakness in Phaseolus vulgaris L. I. Disruption of Development and Hormonal Allocation

A reduced concentration of cytokinins may cause the abnormal growth and development found in F1 hybrids between Andean and Mesoamerican races of Phaseolus vulgaris L. In this study, concentrations of the transportable cytokinin zeatin riboside (ZR) were measured by ELISA for ZR (cross reactivities dihydrozeatin, 14%, zeatin 7.6%) in roots, stems, and leaves of a Phaseolus Mesoamerican landrace (P. vulgaris L. cv. Redkloud), an Andean landrace (P. vulgaris L. cv. Batt), and their F1 hybrids. Concentrations of ZR in roots and leaves of F1 hybrids were significantly less than that found in roots and leaves of parental cultivars. Approximately 90% of the ZR found in F1 hybrids was found sequestered in the stems, whereas cytokinins of the parental cultivars were distributed throughout the plant (roots: Batt 37%, Redkloud, 44%; stems: Batt 35%, Redkloud 42%; leaves: Batt 28%, Redkloud 14%). These results suggest that abnormal growth and development of F1 hybrids may involve interruption of the regulation of cytokinin allocation, thereby disrupting the root-shoot feedback loop between root-sourced cytokinins and putative shoot-produced factors. Received October 15, 1998; accepted May 12, 1999     

Cytokinin Activity in Lupinus albus

The cytokinin content of the root exudate and leaves of fruiting white lupin plants (Lupinus albus L.) was investigated at 2 weekly intervals after anthesis of the lowest flower on the primary inflorescence. Up to 8 weeks after anthesis the level of cytokinins in the root exudate increased. However, at 10 weeks after anthesis insufficient sap was produced for analysis. Cytokinins co-eluting with zeatin and zeatin riboside were detected in the root exudate after fractionation on Sephadex LH-20. The cytokinin levels in the mature leaves steadily increased up to 8 weeks after anthesis and thereafter remained relatively constant. Three peaks of activity, co-eluting with zeatin, zeatin riboside and the glucoside cytokinins were recorded in the leaf extracts. The level of glucoside cytokinins in the leaves was high at 8 and 10 weeks after anthesis. Paper chromatography of extracts of fruits collected at 2 weeks after anthesis indicated that as fruit development proceeded there was a build up of cytokinin in this region of the plant. It is suggested that, in the white lupin, the cytokinins translocated to the shoot are accumulated in the leaves and in the fruits and that it is only later when there is a considerable decrease in sap (10 weeks after anthesis) production that a decreasing supply of cytokinins leads to shoot senescence.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Excretion of cytokinins into the cultivation medium by suspension-cultured tobacco cells

The dynamics of individual cytokinins were determined in both the cells and the cultivation medium during the subculture interval of cell suspension cultures of Nicotiana tabacum L., line VBI-0. The amounts of cytokinins detected in the cultivation medium were less than 1 pmol ml^-1 of suspension. In the late stationary phase, the levels of isopentenyladenosine, as well as that of dihydrozeatin and its riboside, increased significantly. However, when expressed per cell number, the levels of zeatin- and isopentenyladenine-type cytokinins in both the cells and medium were at a maximum at the beginning of the subculture interval and then gradually decreased. Cytokinins were excreted from the cells during the whole subcultivation period, and their concentrations in the cultivation medium were found to be approximately in proportion to their momentary levels inside the cells. The excretion might thus represent one of the mechanisms controlling endogenous cytokinin concentrations.