Effects of temperature on phyllody expression and cytokinin content in floral organs of rose flowers

Formation of leaf-like organs known as phylloids in Rosahybrida cv. Motrea flowers was promoted by exposure of plants toelevated temperatures. At a day/night temperature regime of26°C/21°C respectively theproportion of malformed flowers exhibiting phyllody was four times higher thanthat in flowers of plants grown at21°C/15°C. The number ofpetals in phyllody-expressing flowers was higher than that in normal flowers.The total content of endogenous cytokinins in young flower buds of plantsexposed to the lower temperature was six times higher than that at the highertemperatures. The effects of the reduced temperature were pronounced on all thegroups of cytokinins examined. However, the proportion of the various cytokiningroups remained similar at both temperature regimes. In contrast to thecytokinins in the flower buds, the content of all cytokinin types in youngleaves increased following exposure to the higher temperature and was reducedbythe lower temperatures. After 11 weeks at the lower temperature, about18% of the flowers remained malformed, whereas at the higher temperatureabout 20% of the flowers still remained normal. All thephyllody-exhibiting flowers were formed on vigorously grown basal shootscharacteristic to Rosa hybrida plants, whereas the normalflowers at the elevated temperatures were formed on lateral shoots which weremost distal to the plant base. However, irrespective of the season, thepresenceof normal and malformed flowers was observed on plants kept growing at standardconditions of 30°C/17°C inthegreenhouse. This phenomenon led us to examine the cytokinins in floral organsofnormal and malformed cv. Motrea flowers grown in the greenhouse as well as inflowers of a complete rose mutant known as a 'Green Rose(Rosa chinensis viridiflora). The highest content ofcytokinins was found in the pistils and stamens of normal 'Motreaflowers. On the other hand, the content of cytokinins in leaf-like style-tubesin the malformed flowers as well as in partially malformed ovaries at the baseof phylloids was significantly lower. A low content of cytokinins was alsopresent in petals of both normal and phyllody-exhibiting flowers and the lowestcontent has been found in the phylloids of the 'Green Rose. Apossibility of mutant deviations in metabolism of cytokinins in rose plants isdiscussed.     

Effects of applied auxin,gibberellin and cytokinin on the activity of peroxidases in the peduncles of rose flowers

The activity of peroxidases in the proximal part of the flower peduncle of rose cv. Nubia was promoted by exogenous application of auxin but not by gibberellin or cytokinin. In cv. Mercedes the activity was promoted also by gibberellin and cytokinin. In the distal parts of the peduncles of both cultivars, peroxidase activity was not affected by any of the applied growth regulators. In young flowers of cv. Nubia the protein content of the penduncles was affected only by cytokinin, and in aged flowers only by auxin, while in Mercedes peduncles the content of protein was not affected by any of the applied growth regulators. The specific activity of peroxidases was promoted by auxin in peduncles of Nubia and by both auxin and cytokinin in peduncles of Mercedes flowers.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Stamens and gibberellin in the regulation of corolla pigmentation and growth in Petunia hybrida

Removal of stamens, or even of only the anthers, at an early stage of corolla development, before the start of main anthocyanin production, inhibited both growth and pigmentation of attached corollas of Petunia. When only one or two stamens were removed from one side, the inhibition was restricted to the corolla side adjacent to the detached stamens. Application of gibberellic acid (GA₃) substituted for the stamens in its effect on both growth and pigmentation. In detached corollas, isolated at the early-green stage and grown in vitro in sucrose medium, GA₃ promoted growth and was essential for anthocyanin synthesis. A marked enhancement of anthocyanin production was observed 48 h before the increase in corolla growth rate. Corollas detached at later stages were able to continue their growth and pigmentation in sucrose without GA₃. When Paclobutrazol (-[(4-chlorophenyl)-ethyl]-(1,1-dimethylethyl)-H-1,2,4-triazol-1-ethanol), an inhibitor of gibberellin biosynthesis, was added to the growth medium of in-vitro-grown corollas, pigmentation was inhibited but there was no effect on corolla growth. Low levels of GA₃ counteracted the Paclobutrazol effect on pigmentation but did not affect growth. The above results indicate that the effect of GA₃ (and probably that of the stamens) on corolla growth is independent of its effect on pigmentation. Gibberellic acid and paclobutrazol had no effect on [¹⁴C]sucrose uptake by in-vitro-grown corollas. The activity of phenylalanine ammonialyase was correlated with the effect of stamens and GA₃ on pigmentation in corollas grown in vivo and in vitro.Abbreviations GA gibberellin - GA₃ gibberellic acid - PAC Paclobutrazol - PAL phenylalanine ammonia-lyase     

Morphological development of normal and phyllody expressing Rosa hybrida cv. Motrea flowers

A large number of soybean (Glycine max L. Merr.)flowers and young pods abscise rather than develop into mature pods. Flower andpod drop or abortion accounts for the majority of total reproductive abscissionand influences potential soybean yield. The objectives of this study were todetermine the patterns of flower, pod and seed development under treatmentswiththe growth regulators, 2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP) and6-benzylaminopurine (BAP), applied at the early reproductive stages, and toexamine the association of reproductive abscission with growth characteristicsand agronomic traits, including seed yield and seed weight. Small seeded [cvPungsan (11.1±0.4 g100-seed^–1)] and large seeded [cv Manlee(21.0±0.5 g 100-seed^–1)]genotypes were separately planted in the greenhouse and field, and treated witheither 2,4-DP or BAP. 2,4-DP (a synthetic auxin) and BAP (a syntheticcytokinin)were each applied at three concentrations (i.e. high, intermediate or low):0.12mM, 0.08 mM, 0.04 mM, and 1.5mM, 1 mM, 0.5 mM respectively. High andlow concentrations were employed for greenhouse experiments to examine thenumber of flowers per plant in pots. With the exception of low BAP (0.5mM) treatment in Pungsan, all treatments increased total podnumberwith various numbers of seeds per pod. Low 2,4-DP (0.04 mM) inbothgenotypes or BAP (0.5 mM) in Manlee significantly reduced flowerabortion and delayed abscission of pods in both genotypes, resulting inincreased pod setting rates. Under field conditions using intermediateconcentrations, 1 mM BAP significantly increased 100-seed weightto22.3 g at R1 in Manlee and 11.9 g at R3 in Pungsan.BAP (1 mM) at R3 in Pungsan significantly improved seed yield(40.1g plant^–1). Maturity was not significantlyaffected by either application in Manlee, but was significantly affected by BAPin Pungsan. In Pungsan, 2,4-DP increased pod number, plant height and nodenumber, but decreased 100-seed weight in Pungsan treated at R1, causing nosignificant change of seed yield. This study suggested that exogenousregulatorssignificantly influenced reproductive and growth characteristics, andconsequently seed yield, but increase of pod number was not always beneficialfor seed yield.     

Water relations and growth of rose plants cultured in vitro under various relative humidities

Stomatal malfunctioning is one of the main reasons why plants desiccate when transferred from in vitro to greenhouse conditions. In order to overcome this problem in Rosa hybrida cv. Madame G. Delbard (R) Deladel, two techniques, bottom cooling and water vapour permeable lid, were used. Both methods aimed to increase the vapour pressure gradient between leaf and atmosphere and consequently to improve plant transpiration.The results showed that these techniques increased leaf resistance to dehydration and improved stomatal regulation. Water relations of treated plantlets were similar to those generally observed in hardened plants: lower leaf water and osmotic potentials, and lower leaf water content than in the control ones. Osmotic adjustment occurred in treated plantlets maintaining turgor pressure. Each technique also induced some effects on growth during the rooting phase: with bottom cooling, roots were shorter, with permeable lids, apices were necrosed.These results are discussed in terms of physiological causes and in terms of effect during the following acclimatization.Abbreviations AWC absolute water content - DW dry weight - FW fresh weight     

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 M 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 M 2,4-D and 4.44 M BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Effects of thidiazuron on micropropagation of rose

Summary Rose (Rose hybrida L.) plants were micropropagated by axillary shoot proliferation method. Maximum number of microshoots per shoot tip explant were obtained on MS medium supplemented with 5 to 10µM thidiazuron (TDZ). The microshoots formed rooted plants on MS hormone-free medium. No difference in the rooting of microshoots produced on medium containing TDZ or N⁶-benzyladenine was observed. The regenerated plants were successfully transplanted to the field and appeared similar to the parent plant in morphologic features.     

Factors affectingin vitro establishment and shoot proliferation ofRosa hybrida L. andRosa Chinensis minima

Summary The effects of nodal explants collected at different plastochrones, use of various benzyladenine (BA) concentrations, sources of carbohydrates, and phases of the culture medium on shoot establishment and proliferation ofRosa hybrida L. andR. chinensis minima were evaluated. Higher numbers of shoots per explant were obtained fromR. hybrida cv. Carefree Beauty explants proximal to the apical meristem than those from distal nodes. However, proliferating shoots derived from plastochrones proximal to the apical meristem had a lower number of leaves/explant and were shorter than those derived from other distal plastochrones. Although shoot proliferation increased with higher BA concentration in the medium, a concentration of 4.4 μM BA was found optimum for axillary bud-break and shoot development forR. hybrida cvs. Adelaide Hoodless and Cuthert Grant. A higher shoot proliferation rate was observed forR. hybrida cv. Carefree Beauty explants grown on a medium containing 55.5 mM fructose than 58.4 mM sucrose. However, no differences were observed forR. hybrida cv. Cuthert Grant grown on a medium containing either fructose or sucrose. The mean number of shoots/explant was higher forR. chinensis minima cv. Red Sunblaze explants grown on a liquid (4.5) than on a solid medium (1.7) for the first reculture; while no significant differences between the two phases of the medium were observed for the second reculture. However, a higher mean number of shoots/explant was observed on solid-phase (4.0) than liquid-phase medium (3.4) for the third reculture. A higher mean number of leaves/shoot was obtained on a solidified medium rather than liquid medium in the first two consecutive recultures, while no differences were observed for the third reculture. Although a significant effect of BA concentration on mean number of shoots/explant was observed for Red Sunblaze nodal explants, the influence of BA concentration decreased in the two consecutive cultures for both phases of the medium. Hyperhydricity was observed on Red Sunblaze shoots grown on the liquid-phase medium.     

Genetic and biochemical studies on the conversion of flavanones to dihydroflavonols in flowers of Petunia hybrida

Summary Chemogenetic investigations and precursor experiments on flowers of Petunia hybrida suggest that recessive alleles of the gene An3 block the biosynthetic pathway of flavonols and anthocyanins between the flavanone and dihydroflavonol step. In confirmation of this hypothesis, activity of the enzyme flavanone 3-hydroxylase, which catalyses the conversion of flavanones to dihydroflavonols, was readily demonstrated in enzyme preparations from flowers of lines with the dominant allele An3, whereas no or very low activity could be found in extracts from lines with recessive alleles (an3an3). A second genetic factor is described which clearly reduces the amount of flavonols in the flowers but not the amount of anthocyanins. Crossing experiments revealed that this factor represents a third allele of the An3 gene. It is referred to as an3-1. As expected, a residual flavanone 3-hydroxylase activity of about 10% could be found in enzyme extracts from plants with the an3-1 allele. The decreased level of dihydroflavonol formed under this condition is obviously still sufficient for anthocyanin formation but not for flavonol synthesis.Similar to flavanone 3-hydroxylases from other plants, the enzyme of Petunia is a soluble enzyme and belongs according to its cofactor requirements to the 2-oxoglutarate-dependent dioxygenases. The residual flavanone 3-hydroxylase activity found in plants with the an3-1 allele is identical to the activity extracted from An3-genotypes with regard to cofactors, substrate specificity and most of the inhibitors. The difference observed in the respective pH-optima and the genetic data suggest that the mutation providing the an3-1 phenotype is localized in the structural gene for flavanone 3-hydroxylase.     

Relationship between gibberellin and cytokinin activity and flowering in Rosa damascena Mill.

Kinetin at 10 mg l^–1 increased the number of flowers produced on Rosa damascena plants while GA₃ inhibited flowering. In the leaves of non-flowering plants GA-like activity was high while specific cytokinin activity (fraction-II) was significantly higher in flowering plants. A novel compound 10- methyldihydrozeatin riboside and isopentenyl-adenine were identified from TLC fraction-II while TLC fraction-I yielded zeatin and 2-hydroxy-6-methylaminopurine.Abbreviations TLC thin layer chromatography - BA N⁶-benzyladenine - GA₃ gibberellic acid CIMAP communication No. 92-40J     

Alleviation of frost damage to pear flowers by application of gibberellin

Adverse effects of gibberellin applications on pear trees after frost such as small fruit size, abnormal fruit shape and poor return bloom are often attributed both to the sole use of GA₃ and its overdose. It is unclear whether protection against spring frosts before flower opening is more efficient when GA is applied directly after frost, i.e. before flower opening, or at full bloom or both. In April 2003, early spring frosts at Klein-Altendorf near Bonn, Germany damaged ca. 88% flowers of the early flowering cv. Alexander Lucas, 64% of cv. Conference and ca. 25% of flowers of the later flowering cv. Comice pears. Hence, the objective of the present work was to investigate the optimum timing of the application of low doses of the combined GA₃ and GA_{4 + 7} to improve parthenocarpic fruit set in pears, while maximising fruit quality and size for trees affected by a severe spring frost before full bloom. Return bloom was also considered and quantified. Frost-affected pear trees were treated with gibberellin GA₃+GA_{4 + 7}, either immediately after the frost, at the white bud stage, or at full bloom or both to improve parthenocarpic set. Early flowering cv. Alexander Lucas pear was most affected by the early spring frost, but lost only 25% of fruitlets at June drop, irrespective of GA treatment. June drop was, however, severe in the two other cultivars least affected by frost, i.e. by 33% in cv. Conference and 55% in cv. Comice. Both initial and final fruit set were significantly increased by a combined application of GA₃+GA_{4 + 7} at full bloom, without affecting return bloom, but June drop was also enhanced by GA application. The largest positive effect of GA application on fruit yield, an additional 2 kg of fruit per tree equivalent to €1200/ha, was apparent with the cv. Alexander Lucas, i.e. the cultivar most affected by frost. There was no loss in fruit quality viz fruit size after any of the GA applications with any of the pear cultivars examined and no increase in abnormally-shaped, elongated fruit.     

Endogenous cytokinins in rose petals and the effect of exogenously applied cytokinins on flower senescence

In extracts from rose petals cytokinin activity was detected by Amaranthus bioassay in HPLC eluates corresponding to the standards: Z, ZR, 2iP and 2iPA; subsequently, the presence of two groups of endogenous cytokinins was confirmed by ELISA.Measurements of senesence indicators (cell sap osmolarity and conductivity) and observations of flower vase-life indicated that when the above cytokinins were applied as holding solutions they delayed flower senescence by 34–56% and prolonged rose longevity.Abbreviations B.H.T. 2.6-di-t-buytl-4-methyl phenol - ELISA Enzyme linked Immunosorbent Assay - HPLC High Performance Liquid Chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - Z trans-zeatin - ZR trans-zeatin riboside     

Influence of season on rose pollen quality

Summary In vitro tests of pollen germination were carried out at different periods during an annual cycle in order to study environmental influence on the quality of Rosa hybrida L. pollen during its maturation process. This quality was evaluated by taking into account the rate of germination as well as the average length of emitted pollen tubes. In addition, during an annual hybridization period, a few pollinations were carried out in vivo with pollen of the same origin in order to study the evolution of the fertilization results, as attested by number of achenes per resulting hips. During the period covered by the experiments, the evolution of the pollen quality detected in vitro can be related to that of seed setting success. Of the two criteria used in vitro to evaluate pollen quality, the factor most liable to influence in vivo fertilization success seems to be the average length of emitted pollen tubes.     

Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.     

Leaf water and carbohydrate status of VA mycorrhizal rose exposed to drought stress

Summary Shoot water relations and carbohydrate levels were compared for droughted nonmycorrhizal and vesicular-arbuscular (VA) mycorrhizalRosa hybrida L. cv ‘Samantha’ plants grown with high and low phosphorus fertilization. Leaf diffusive conductance (g _i ) of plants colonized byGlomus intraradices Schenk and Smith andGlomus deserticola Trappe, Bloss and Menge were 2 × and 1.5× greater, respectively, than in nonmycorrhizal plants. Regardless of P fertilization, leaf osmotic and bulk water potentials were 0.5 to 1.1 MPa higher in mycorrhizal than in nonmycorrhizal plants. Leaf starch, chlorophyll and water contents while fructose, glucose and total soluble carbohydrates were lower. Level of P fertilization had no effect on water relations or soluble carbohydrate content of nonmycorrhizal roses. The water status of droughted rose was impoved more byG. intraradices than byG. deserticola. Washington State University College of Agriculture and Home Economics Research Center Scientific Paper No. 7375.     

Pollen tube expression of pseudo-self-compatibility (PSC) inPetunia hybrida

Summary AnS _1.1 self-incompatible (SI) petunia plant which showed atypical seed set was found in an I₇ population. This plant showed a strong SI reaction when selfed but produced varying amounts of seed when used as the seed parent in crosses with unrelated individuals homozygous for the sameS allele. Reciprocal crosses yielded no seed indicating that the reaction was a stylar response. Self seed obtained by high temperature treatments produced 18 plants, all of which exhibited the parental characteristics, the ability to reject self pollen but accept, to varying degrees, pollen bearing the sameS allele from unrelated plants. Several petunias homozygous forS ₁, and exhibiting various levels of PSC as determined by self seed set, progeny tests and temperature treatments, were used as pollen parents. The mean seed set of these crosses produced a ranking of the pollen parents which reflected the PSC levels obtained by other methods. The behavior of the F₁ and F₂ populations suggests that the pollen discriminating ability may be a simply inherited, dominant character in these plants. The styles of these unusual petunias illustrate the participation of the pollen tube in determining PSC.Scientific Journal Series Paper Number 10.479 of the Minnesota Agricultural Experiment Station     

Phytotoxicity of tracer 120SC on greenhouse-grown rose (Rosa sp.) flowers

Tracer 120SC is effective against several insect pests of different crops, but it may be phytotoxic. The objective of this study was to quantify the phytotoxic effect of Tracer 120SC on rose. Phtotoxicity of Tracer 120SC was tested at three rates. A single application of Tracer 120SC caused acute phytotoxic damage to leaves, shoots and flower stalks at all tested rates. Two to three days after spraying symptoms were observed on 32.2 to 52.0% of sprayed shoots, with severity of 26 to 38%. Damage on individual shoots without flower buds varied between 21.1 and 37.6%. Similarly, the proportion of damaged leaves per flower stalk ranged from 57.8 to 62.3%. Flower damage was highest of any tissue, with 79.5 to 90% of tissue exhibiting phytotoxic symptoms. Taken together all flower stalks exhibited damage and were not marketable.