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Mid-infrared ATR FT-IR microspectroscopy is applied in conjunction with multivariate statistical analysis on petal samples of Petunia hybrida from wild-type and from two transgenic lines in which the PhEXPA1 expansin gene expression was down-regulated and up-regulated, respectively. New insights are given on the role of expansin in the rearrangement of the cell wall polymer network. Measurements are done without any previous sample treatment to preserve the native cell wall structure and natural biological variability. An original combination of data analysis techniques is proposed to highlight molecular processes in plant cells, based on an automated spectra selection procedure, Principal Component Analysis, Wilcoxon rank sum test and heat map data representation.

We found that the corolla of petunia ( Petunia hybrida Vilm.) could be conspicuously enlarged by the separate application of three cytokinins: forchlorfenuron (CPPU), N 6 -benzylaminopurine (BA), and zeatin. To obtain the same enlargement as that achieved by CPPU, approximately 30 and 900 times the concentration of BA and zeatin, respectively, were required. CPPU at 3.2 μmol/L increased the limb area of the corollas of 15 cultivars to between 1.3 and 2.4 times (1.8 times on average) the size of the control area. The increase was negatively correlated ( R = 0.58) with the “genetic” limb area (i.e., that of the untreated plant). The enlargement of the corolla caused by cytokinin application was mainly attributed to an increase in cell number in most cultivars. This increase resulted from a high rate of cell proliferation and from prolongation of the cell proliferation phase during corolla development. This anatomical change caused by cytokinin application was similar to the anatomical difference among cultivars because genetic differences in limb area resulted mainly from differences in cell number.

Two novel diacylated anthocyanins and two known monoacylated anthocyanins were isolated from violet petals of Petunia hybrida cv. Surfinia Violet Mini. Their structures were elucidated by spectral methods. The major novel pigment was identified as malvidin 3- O -(6- O -(4- O -(4- O -(6- O -caffeoyl- β -d-glucopyranosyl)- p -coumaroyl)- α -l-rhamnosyl)- β -d-glucopyranoside)-5- O - β -d-glucopyranoside and the minor novel pigment was determined as malvidin 3- O -(6- O -(4- O -(4- O -(6- O -caffeoyl- β -d-glucopyranosyl)-caffeoyl)- α -l-rhamnosyl)- β -d-glucopyranoside)-5- O - β - d-glucopyranoside.

The action of metal ions on detached flowers of Petunia hybrida was investigated to determine how they affected ethylene-promoted senescence. Salts of various metals were applied singly or in combination by 5 s immersion of excised flowers which were then incubated in 1μl l−1 ethylene for 24 h. Of the metals tested, only Ag+ prevented ethylene-induced senescence. Cu2+ and Cd2+ completely, and Hg2+ and Fe2+ partially reversed the effect of Ag+. K+, Na+, Ca2+, Mg2+, Al3+, Co2+, Mn2+ and Zn2+ were ineffective. Cu2+ promoted senescence even when applied after Ag+ and a 24-h ethylene treatment. Cu2+ promoted ethylene synthesis, so that senescence was not affected by ethylene treatment unless flowers had been pre-treated with aminoethoxyvinyl-glycine. Ag+ inhibited the promotion of respiration and ethylene production by ethylene treatment of corolla tissue. Cu2+ treatment restored these ethylene effects in Ag+-treated tissue. The action of Cu2+ suggests that ethylene signal perception occurred in Ag+-treated corolla tissue.

An assay of chalcone synthase activity based upon analysis by HPLC has been developed. The method is specific (identification of the product formed), f

Summary Purified protoplasts from Petunia hybrida reduced 2,3,5-triphenyl tetrazolium chloride (TTC), whereas cellular or subcellular debris gave a negative TTC test. The TTC reduction was decreased with increasing concentration of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and iodoacetate (IA). Consequently it could be correlated with plating efficiency. The most formazan was produced by the protoplasts from top-developing leaves. FDA staining could not detect the difference in viability of protoplast isolated from different growth stages of leaves. The TTC reduction test described here offers a colorimetric estimation for the potential of protoplasts to form colonies.

Three sucrose ester types were isolated and identified from the leaf surface lipids of Petunia hybrida. They contained both unbranched and branched fatty acids (from C2 to C8). Further, one of these sucrose esters contained malonic acids. The structures of the sucrose esters were 2,3,4-O- tri-acyl -α- d -glucopyranosyl -1-O- malonyl -4-O- acyl -6-O- acetyl -β- d -fructofuranoside , 2,3,4-O- tri-acyl -α- d -glucopyranosyl -4-O- acyl -6-O- acetyl -β- d -fructofuranoside and 2,3,4,6-O- tetra-acyl -α- d -glucopyranosyl -β- d -fructofuranoside .

Non-disjunction events, spontaneous or induced, are rather frequent in plants. In Petunia, no significant effect of γ-radiations was detected. However, the study of some monogametic mutants characterized by a terminal deletion showed that, at the level of the chromosome pair involved by this deletion, both the frequency of specific trisomics and the recombination fractions between 4 marker loci were considerably increased in the progeny of the mutants as compared with that of the control. These features, i.e. deletion-induced non-disjunction and recombination, may be considered as indirect effects of radiations. Generalization to other species may be of importance in the fields of mammalian genetics and plant breeding.

Summary Nuclei were prepared from isolated mesophyll protoplasts of Petunia hybrida by treatment with triton or according to Blobel-Potter , and incubated with [ 3 H]-labelled double stranded E. coli DNA. Thereafter, the nuclei were repeatedly washed, treated with triton, sedimented with high speed sucrose gradient centrifugation, treated with DNAse, and incubated with unlabelled competitor DNA, respectively. The results demonstrated the presence of E. coli DNA very closely associated with the nuclei. Wether this DNA was only fixed superficially in a stable form or taken up into the nuclei or both of them, could not be decided. DNase incubation under very mild conditions showed a parallel degradation of the fed E. coli DNA and nuclear DNA. This would be consistent with an uptake, but gives no unequivocal evidence.

Summary Three cultivars of Petunia hybrida Vilm. , of differing ozone visible injury sensitivity, were exposed to 40 parts per hundred million (pphm) ozone and/or 80 pphm SO 2 for 4 h to study the relationships of leaf water potential, pollutant exposure, and cultivar sensitivity. Ozone substantially decreased leaf water potential in cv White Cascade but not in cv Capri or White Magic. Sulphur dioxide did not affect leaf water potential but delayed ozone-induced changes. Cultivar sensitivity to ozone-induced changes in leaf water potential was not related to cultivar sensitivity to ozone-induced visible injury.

Seven new steroids have been isolated from leaves and stems of Petunia hybrida . They are 30-hydroxy petuniasterone A (C-30 epimeric mixture), 1α-acetoxy-1,2-dihydropetuniasterone A (designated petuniasterone E), (22 R ,24 S )-7α,24-dihydroxy-22,25-oxidoergosta-1,4-dien-3-one (petuniasterone F), C-24 epimers of (22 R )-7α,22,24,25-tetrahydroxyergosta-1,4-dien-3-one (petuniasterones G 1 and G 2 and C-24 epimers of (22 R )-1-α-acetoxy-7α,22,24,25-tetrahydroxyergost-4-en-3-one (petuniasterones H 1 and H 2 ). The compounds which have 1-α-acetoxy groups may easily be converted into the corresponding A-ring dienes by mild base. The orthoesters and other side-chain variants may be formed from the 24,25-epoxy compounds which co-occur in the plant.

Zusammenfassung Zusammenfassung—Die Wirkung zweier Chalkone auf die Anthocyansynthese in isolierten Petalen von Petunia hybrida wurde untersucht. Dabei zeigte sowohl das 3,4,2′,4′,6′-Penta-hydroxy- als auch das 4,2′,4′,6′-Tetra-hydroxy-3-methoxy-chalkon-4′-glukosid in Gegenwart von D-Glukose-[1- 14 C] eine substitutionsspezifische Wirkung. Es wird stets die Synthese desjenigen Anthocyans gefordert, das dem eingesetzten Chalkon in der Substitution entspricht. Dies last sich kaum anders als damit erklaren, das die Chalkone zur Synthese der Anthocyane herangezogen werden. Bei der gemeinsamen Futterung von Chalkon und Acetat-[1- 14 C] zeigt nur das 3,4,2′,4′,6′-Pentahydroxy-chalkon-4′-glukosid eine substitutionsspezifische Wirkung.

The effects of N and Ca nutrition on plant growth and shoot elemental content of Petunia × hybrida Hort. Vilm. - Andr. ‘Coral Sea’ were evaluated. Nitrogen and Ca were applied separately or in combination in three experiments: (1) N at 0, 100, 200 or 400 mg l−1; (2) Ca at 0, 75, 150 or 300 mg l−1; (3) N at 0 or 100 mg l−1 and Ca at 0 or 150 mg l−1 combined factorially. Shoot and root dry weights, branch length and flower number were highest when plants received 100 mg l−1 N. Plants treated with 150 mg l−1 Ca had the highest shoot and root dry weights. Branch length was maximal at 300 mg l−1 Ca. Nitrogen and Ca interacted to increase shoot dry weights, branch number and length, leaf area and flower number. Increasing N concentrations increased N and decreased P, Mn and Zn shoot contents. Calcium content of shoots increased while N, P and Mg decreased in response to increasing applications of Ca to petunia plants. Minimal N and Ca tissue concentrations for optimal P. × hybrida growth were 3.3 and 0.67%, respectively.

Haploid and diploid protoplasts of the two Solanaceous species Datura innoxia Mill. and Petunia hybridia L. were exposed to two different mutagens, increased doses of X-rays, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). With both species the survival rates of haploid protoplasts decreased exponentially with increased doses of X-rays and increased concentrations of MNNG. Diploid protoplasts showed a higher resistance than haploids only at higher mutagen doses or concentrations. After the MNNG-treatment of haploid protoplasts from Datura innoxia, four mutants with altered pigment patterns were isolated.

Summary Experiments with FITC-Iabeled bovine-serum-albumin, with FITC-labeled and nonlabeled bacteriophage lambda have shown, that proteins and bacteriophage can be taken up by swelling and germinating pollen grains of Petunia hybrida. Following reisolation of the phage from the pollen we were able to demonstrate biological activity in a small fraction of the phage.

Fraction 1 protein from five species of Petunia and selected genetic lines and cultivars of Petunia hybrida Vilm. has been isolated and purified using a specific immunoabsorbent. Isoelectric focusing of the S-carboxymethylated protein in the presence of 8 M urea resolved three large subunit polypeptides with identical isoelectric focusing patterns for all five species and the P. hybrida Vilm. cultivars and lines. Two small subunit polypeptides are commonly shared by P. axillaris, P. hybrida, P. inflata, P. parodii and P. violacea, whilst the species P. parviflora had a single polypeptide located between the two polypeptides found in the other species and cultivars. The electrofocusing pattern of the small subunit polypeptides confirmed the established taxonomic relationships among these Petunia species based on breeding behaviour, chromosome number, morphology and biochemical analyses.

Summary Isolated mesophyll protoplasts of Petunia hybrida were incubated with phages. Optimal conditions for the incubation with phages were obtained with a "washing diffusion" procedure of protoplasts plated in agar blocks. Auto radio graphical and EM studies revealed an uptake of phages into the cytoplasm of the protoplasts.

3,4,2′,4′,6′-Pentahydroxychalcone 4′-glucoside, which is specific for B-ring substitution, triggers an incorporation of [1- 14 C]acetate into cyanidin 3-monoglucoside in Petunia hybrida , at a stage when anthocyanin accumulation is linear and intensive. This incorporation could not be specifically prevented by adding either transcriptive or translational antimetabolites. Sodium diethyldithiocarbaminate, which specifically complexes with Cu 2+ ions, produces the same effect. This similarity in action suggests that both reagents affect those enzymes of the anthocyanin pathway which require bivalent metal ions as co-factors.

Three methods of selection are described for the production of somatic hybrids of Petunia hybrida and Petunia parodii . Using cultured leaf protoplasts of the F 1 sexual hybrid, and the two parents, it was possible to develop media in which only protoplasts of the sexual hybrid would grow. Following fusion treatments, protoplasts were plated in such media in order to assess the frequency of somatic hybrid plant production and selection efficiency.

Seeds of ‘Commanche’ petunia (Petunia hybrida Vilm.) were germinated (GE) or imbibed (IM) on the surface of Laponite 445 or Natrosol 250 HHR gels prepared from 0, 0.5 or 1.0-fold Hoagland's solution. These seeds and dry seed (DS) were incorporated in the gels and extruded onto peat—vermiculite. When compared to a dry-seed control (without gels), DS in either gel without nutrient additive did not increase percentage of transplantable seedlings, but IM and GE resulted in a 32% increase. In comparing only fluid-drilled treatments, percentage of transplantable seedlings was increased by GE relative to IM in both gels, but by IM relative to DS only in Laponite. In both gels, emergence rate (T50) and uniformity (T10–90) and shoot fresh weight were influenced in the order GE > IM > DS. Increasing nutrient concentration in both gels decreased T10–90 but had no effect on T50 or shoot fresh weight. Percentage of transplantable seedlings was increased in Laponite but decreased in Natrosol with increasing gel nutrient concentration. The highest percentage of transplantable seedlings was achieved with GE in Natrosol without nutrient additive (75.4%) and in Laponite with the highest nutrient concentration (81%), each giving approximately one-half of the T50 and T10–90 values of the dry-seed control which yielded 53% transplantable seedlings.

The incorporation of shikimate-[ 14 C] and cinnamate-[ 14 C] into 3′,4′- and 3′,4′,5′-hydroxy substituted anthocyanins was studied in isolated petals of Petunia hybrida . According to the dilution values, the incorporation of shikimate-[ 14 C] was about 3–6 times better than that of cinnamate-[ 14 C]. However a comparison of the incorporation of the 2 precursors on a relative basis showed no significant differences in the relative proportions of the specific activities of the 3′,4′-dihydroxysubstituted cyanidin-3-monoglucoside and the 3′,4′,5′-trihydroxysubstituted delphinidin-3-monoglucoside. This result and the [ 14 C]-incorporation behaviour of the 3′-methoxy-4′-hydroxysubstituted peonidin-3-monoglucoside do not support the hypothesis that there are alternative pathways of flavonoid biosynthesis.

Summary The effect of cold treatment was studied on the induction of pollen embryoids in Petunia hybrida. Anthers containing uninucleate pollen grains were kept at 6°C for 48 hrs and then cultured on NITSCH'S medium supplemented with 10-6 M kinetin or 15 % coconut milk. In the kinetin containing medium the percentage of anthers producing embryoids was more than double, compared to the anthers inoculated in the same medium but without cold treatment. In the coconut milk medium, anthers often callused but cold treatment was found to suppress callusing and to lead large numbers of pollen grains directly towards the path of embryoid development.

Summary 4400 anthers of two pure lines of Petunia hybrida and of the hybrid between them cultivated in vitro produced 1 haploid and 31 diploid or triploid plants. As shown by the use of the hybrid anthers with anthocyanins marker genes all these different levels of ploidie are derived from the pollen material.

Summary Earlier experiments have established that chalcones are natural intermediates in the biosynthetic pathway of flavonoids. But there was no knowledge about their degree of gly-cosidation. Therefore we synthesized 3,4,2′,4′,6′-penta-hydroxy- and 4,2′,4′,6′-tetra-hydroxy-3-methoxy-chalcone-4′-glucoside and used these chalcones as carriers in trapping experiments. In this way we ascertained the existence of glucosidiced 3,4,2′,4′,6′-penta-hydroxy-chalcone in the biosynthetic pathway of flavonoids in petals of Petunia hybrida at the stage of intensive and linear anthocyanin accumulation.

An albino complementation selection procedure has been developed for the selection of somatic hybrids of Petunia hybrida and Petunia parodii . The method involves fusing albino suspension culture protoplasts with leaf mesophyll protoplasts of the other species, and selecting green colonies formed as a result of complementation and selective growth. This selection procedure should be readily applicable to a wide range of inter-species fusions, including those of sexually incompatible species.

From leaves of Petunia parodii were isolated four epoxy group-bearing petuniasterones. These were shown to be (22 R ,24 R )-7α-acetoxy-4β,5β-epoxy-17β,22,24,25-tetrahydroxyergost-1-en-3-one 22,24,25-[(methylthio)carbonyl]orthoacetate (petuniasterone I); (22 R ,24 R )-7α,12α-diacetoxy-4β,5β-epoxy-22,24,25-trihydroxyergost-1-en-3-one 22,24,25-orthoacetate (petuniasterone J); (22 R ,24 R )-7α-acetoxy-16β,17β-epoxy-22,24,25-trihydroxyergosta-1,4-dien-3-one 22,24,25-orthoacetate (petuniasterone K); and (22 R ,24 R )-7α-acetoxy-16β,17β-epoxy-22,24,25-trihydroxy-ergosta-1,4-dien-3-one 22,24,25-[(methylthio)carbonyl]orthoacetate (petuniasterone L). From leaves of Petunia integrifolia were isolated (22 R ,24 R )-7α,12ξ-diacetoxy-11β,22,24,25-tetrahydroxyergosta-1,4-diene-3-one 22,24,25-orthoacetate (12ξ-acetoxy-11-β-hydroxypetuniasterone D 7-acetate); (22 R ,24 R )-7α,12ξ-diacetoxy-11β,22,24,25-tetrahydroxyergosta-1,4-dien-3-one 22,24,25-orthopropionate (12ξ-acetoxy-11β-hydroxypetuniasterone M 7-acetate); (22 R ,24 R )-7α,22,24,25-tetrahydroxyergosta-1,4-dien-3-one 22,24,25-orthopropionate (petuniasterone M); and (22 R , 24 R )-12α-acetoxy-7α,22,24,25-tetrahydroxyergosta-1,4-diene-3-one 22,24,25-orthopropionate (12α-acetoxypetuniasterone M). From leaves of Petunia hybrida were isolated 30-hydroxypetuniasterone A 7-acetate and petuniasterone C 7-acetate.

A 3′, 4′-dihydroxy or a 3′, 4′, 5′-trihydroxy substitution pattern of dihydroflavonols is required for their conversion into the corresponding anthocyanins in a white flower of Petunia hybrida . The presence of a 5-hydroxyl group is not required. B-ring methylated dihydroflavonols were not converted into the corresponding anthocyanins. In case of a 4′-methoxy substituted dihydroflavonol a 4′-hydroxyanthocyanin is obtained, suggesting demethylation of this compound. The conversion of synthetic (±)- trans -2,3-dihydroflavonols into anthocyanins proceeded almost as well as with natural compounds. The results demonstrate that the cinnamic acid starter hypothesis for the origin of B-ring substituents is not correct for B-ring methylation.

Summary In corollas of Petunia hybrida the presence of a glucosyltransferase capable of catalyzing the transfer of glucose from UDP-glucose to the 3-0-position of cyanidin was demonstrated. The enzyme could also use delphinidin as a substrate. The glucosyltransferase exhibited a pH optimum between 7.5-8.0 and had a «true Km« value of 1.7 mM for UDP-glucose. For the substrates cyanidin and delphinidin allosteric behaviour was observed. The Hill number was determined to be 1.9 for cyanidin. The K' values were 2.5 × 10 -3 mM for cyanidin and 5 × 10 -3 mM for delphinidin. Cyanidin 3-glucosylation was inhibited by delphinidin and delphinidin 3-glucosylation was inhibited by cyanidin, either inhibition being of the uncompetitive type. The enzymatic activity was controlled by the genes An1 and An2. Genotypes in which either of these genes was in the homozygous recessive state showed 20% of the activity of the control value, without changes in the substrate affinities of the enzyme.

Summary The uptake of 3 H-labelled E. coli DNA into isolated mesophyll protoplasts of Petunia hybrida was studied under sterile conditions. Following the incubation with bacterial DNA, nuclei were prepared from the protoplasts by a treatment with Triton X-100 and their DNA was extracted. Using CsCl density gradient centrifugation bacterial DNA could be recovered in a high molecular albeit in a somewhat degraded state. The efficacy of poly-(L-lysine) is consistent with an uptake of the exogenous DNA, at least into the cytoplasm of the protoplasts: The DNA recovered from the nuclei was equivalent to 10 4 molecules of the fed DNA in the absence of poly-(L-lysine) resp. to 10 5 molecules in its presence. Experiments under non sterile conditions may lead to erraneous results due to an uptake of bacteria with labelled DNA into the protoplasts.

Phytic acid has been identified by paper electrophoresis and NMR spectroscopy in extracts of pollen from Petunia hybrida. In this species pollen wa

The ethylene releasing growth regulator ethrel (2-chloroethyl) phosphonic acid, in liquid and 5% granulated dry slow-release forms, was applied to petunias cultivars ‘Royal Cascade’, ‘Chiffon Cascade’, ‘White Cascade’ and ‘Pink Cascade’ as foliar spray, soil drenches or mixed in the soil medium. Granular and liquid forms of ethrel were effective as growth retardants and branch-including agents. Plant height and diameter were reduced and up to 300% increase in basal branches occurred, compared with control plants. The most effective and consistent results were observed with ethrel used as a soil drench. Best treatments for height control without phytotoxic symptoms were 250–500 p.p.m. as soil drenches, 500 p.p.m. foliar sprays and 0.1–0.4 g/pot soil incorporated granulated ethrel formulation. Branches forced by ethrel treatment remained functional. Flowers were delayed by ethrel treatments, but treated plants transplanted outdoors produced more flowers during the growing-season.

Summary Three to seven week old sterile grown Petunia hybrida plants were associated in vitro with Rhizobium sp. 32H1. Nitrogenase activity could be detected, and polar attachment of the rhizobia to the plant root surface was observed.

Summary Induction of androgenesis without any nutrient medium, is being reported here for the first time. Cold-treatment of buds (0 ± 1 ‰C for 5 days) followed by 1 day at 12 ± 1 °C or 25 ± 1 °C have been observed to induce mitotic activity in Petunia hybrida pollen.

It is shown that the transfer of cytoplasmic genetic information from one line of Petunia hybrida to another has been achieved by protoplast fusion. The method used here has yielded more than 50% of plants regenerated from treated cultures originating from fused heterotypic protoplasts. Some individuals, with unchanged expression of nuclear genes, but transformed from male sterile to male fertile, show that a transfer can involve cytoplasmic genes exclusively.

Summary Protoplasts of Vicia faba L. were isolated from tissue of the shoot apex and from leaves of various ages. They regenerated to calluses in media KM ( Kao and Michayluk, 1975 ) and V-KM, composed of the inorganic salts of V-47 ( Binding, 1974 a ) and the organic ingredients of KM. Important factors were low titres of the Vicia protoplasts and temperatures of 22-26°C. Highest plating efficiencies were achieved if less than 3 · 10 3 protoplasts per ml of Vicia faba were co-cultured with 0.5-1.0 · 10 4 protoplasts of Petunia hybrida. The propagation of regenerated calluses was possible on media MS ( Murashige and Skoog, 1962 ), NN ( Nitsch and Nitsch, 1969 ) and NT ( Nagata and Takebe, 1971 ) if the concentrations of cytokinins did not exceed 1µM.

Summary Excised stem and leaf explants of Petunia inflata and two varieties of Petunia hybrida, „rose du ciel“ and „cascade“ were grown on a defined nutrient medium. The morphogenetic responses of the expiants varied with the growth substances added to the medium. There was either the direct regeneration of shoot buds or the development of a friable callus which differentiated into embryos. Plantlets were produced from the embryos.

Summary In the delphinidin-genotype of Petunia hybrida the influence of the light intensity on the accumulation of the 3-monoglucosides of delphinidin, petunidin, and malvidin was measured. In the intensity range tested, in white light there is an increase in the accumulation of all 3 anthocyanins with higher illumination; in red no intensity dependency is observed; in far-red and in blue with higher intensities the accumulation of delphinidin-3-monoglucoside is specifically lowered, whereas the accumulation of petunidin- and malvidin-3-monoglucoside remains about the same. It is concluded that there is a difference in the intensity dependency of the accumulation of the 3 anthocyanins with wavelength.

Summary Protoplasts isolated from the mesophyll of some pure lines of Petunia hybrida are easily coloured by FITC (fig. 1). FITC has no harmful effect. On the contrary, in some cases protoplast division and callus formation was stimulated (fig. 2). So FITC could be useful in marking protoplasts in somatic hybridization experiments. Also FITC-labelled bovine serum albumin was taken up by the protoplasts (fig. 3). Disc electrophoresis of the proteins extracted from aceton powder of protoplasts incubated with the FITC-serum albumin demonstrated that not split off FITC, but indeed intact FITC-serum albumin was incorporated. At the highest concentration of FITC bovine serum albumin poly-L-lysine has a stimulating effect on this uptake (fig. 4).

Summary Tracer experiments have established, that chalcones are precursors in the biosynthetic pathway of flavonoids. We synthesized 2′,4′6′,3,4-penta-hydroxy-chalcone and worked out a method to isolate chalcones. By using the penta-hydroxy-chalcone as carrier in trapping experiments, we prouved the existence of chalcones/flavanones in petals of Petunia hybrida in the stage of intensive and linear anthocyanin synthesis. The results support the hypothesis, that chalcone are not only precursors, but also natural intermediates in flavonoid biosynthesis.

Cybrids with nuclei of Nicotiana tabacum and chloroplasts from Petunia hybrida in general accumulate higher amounts of nicotine and (1 S ,2 E ,4 R ,6 R ,7 E ,11 E )-and(1 S ,2 E ,4 S ,6 R ,7 E ,11 E )-2,7,11-cembratriene-4,6-diol than do ordinary tobacco plants. The composition of the cembranic diterpenoids present in the cuticular wax of the cybrid leaves, was, with one exception, found to be similar to that encountered in the leaves of N. tabacum .

Protoplasts have been isolated from leaves of plantlets of haploid Petunia hybrida grown aseptically. In suitable culture media, the protoplasts divided at a high frequency forming cell clusters after 2 weeks.

Summary Isolated chloroplasts of Petunia hybrida var. Cyatype have been transplanted into albino protoplasts isolated from an extrachromosomal inherited variegated Petunia hybrida.

Summary Isolated nuclei of Petunia hybrida have been transplanted into isolated protoplasts of Petunia hybrida , Nicotiana glauca , and Zea mays .

The incorporation of phenylalanine-[ 14 C] into anthocyanins of petals of Petunia hybrida is greater than that of cinnamic acid-[ 14 C]. Moreover, there is a preferential incorporation of phenylalanine-[ 14 C] into delphinidin 3-monoglucoside, as compared with the incorporation into cyanidin and peonidin 3-monoglucosides.