Robert J. Griesbach

Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae)

Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.

TRANSGENE INACTIVATION IN PETUNIA HYBRIDA IS INFLUENCED BY THE PROPERTIES OF THE FOREIGN GENE

Petunia mutant RL01 was transformed with maizeA1 and gerberagdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The sameAgrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, theA1 andgdfr transformants showed clearly different phenotypes. The flowers of the primaryA1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of thegdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in theA1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In thegdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while thegdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonousgdfr cDNA with a lower GC content and fewer possible methylation sites is more ‘compatible’ the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.

Effects of Anthocyanin and Carotenoid Combinations on Foliage and Immature Fruit Color of Capsicum annuum L.

Shades ranging from violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. High-performance liquid chromatography and mass spectrometry analysis of violet and black fruit tissue identified a single anthocyanin that was determined to be delphinidin-3-p-coumaroyl-rutinoside-5-glucoside. Leaf tissue of a black-pigmented foliage genotype contained the same anthocyanin found in fruit but at a considerably higher concentration in comparison to violet and black fruit tissue. Fruit chlorophyll concentration was approximately 14-fold higher in black fruit in comparison to violet fruit that contained relatively little chlorophyll. Beta-carotene, lutein, violaxanthin, and neoxanthin carotenoid concentrations in black fruit were also significantly greater in comparison to violet fruit. High concentrations of delphinidin in combination with chlorophyll and accessory carotenoid pigments produced the characteristic black pigmentation observed in fruits and leaves of selected genotypes. Anthocyanins were accumulated in the outer mesocarp of violet and black fruit and in the palisade and mesophyll cells of black leaves. Consistent with chlorophyll content of respective genotypes, chloroplast density was greater in cells of black fruits. Utilizing Capsicum pigment variants, we determine the biochemical factors responsible for violet versus black-pigmented pepper tissue in the context of described pepper color genes.

Cloning of a cDNA encoding the cranberry dihydroflavonol-4-reductase (DFR) and expression in transgenic tobacco

Abstract A clone representing a fragment of the dihydroflavonol-4-reductase ( DFR ) gene from cranberry was isolated from a genomic DNA library using the tomato DFR gene as a probe. Sequence analysis of the clone confirmed homology to published DFR gene sequences. 3′ and 5′ RACE (rapid amplification of cDNA ends) reactions from cranberry leaf total RNA were used to obtain the entire cDNA sequence. The sequence information was used to amplify a full-length clone by RT-PCR. Sequencing analysis to confirm the identity of the full-length DFR cDNA identified a putative second allele. Segregation analysis suggested that the two sequences are not allelic, but multi-locus. Nucleotide sequence homology of the full-length clones was highest to published DFR sequence from Camellia sinensis (about 80% identity) followed by Forsythia x intermedia , Antirrhinum majus , Rosa hybrida and Petunia hybrida . When expressed using the CaMV 35S promoter, the corolla of flowers of transgenic tobacco plants were much darker pink than the controls. Some flower parts not normally highly pigmented, such as the filaments, were also dark pink. These data confirm the identity and function of the cranberry clones and further suggest that overexpression of the cranberry DFR could be used to increase anthocyanin production in transgenic plants.

The use of indoleacetylamino acids in the in vitro propagation of Phalaenopsis orchids

Abstract Phalaenopsis orchids can be propagated through in vitro culture of inflorescence nodal buds. A procedure was developed whereby nodal buds were induced to grow into shoots. The basal section of the shoots was cut off and placed on a medium which induced formation of protocorm-like structures. The effects of 4 indoleacetylamino acids on proliferation of these structures were examined.

Characterization of the flavonol glycosides in Petunia.

Abstract The flavonol glycosides from 17 different Petunia hybrida cultivars and species were characterized. Fourteen different flavonol glucosides were identified. Eight of these glucosides have not previously been reported for Petunia . They are quercetin and kaempferol 3-glucoside, 3,7-diglucoside, 3-caffeoylsophoroside and 3-caffeoylsophoryl-7-glucoside. A tentative flavonol glucoside biosynthetic pathway is proposed.

1-aminocyclopropane-1-carboxylic-acid-dependent ethylene production during re-formation of vacuoles in evacuolated protoplasts of Petunia hybrida

SummaryEthylene formation from 1-aminocycloprane-1-carboxylic acid (ACC) was studied in whole protoplasts, evaluolated protoplasts and isolated vacuoles from mesophyll cells of Petunia hybrida L. cv. Pink Magic. The re-formation of the large, central vacuole in evacuolated protoplasts and morphological characteristics of both types of protoplasts were examined by electron microscopy. Both the normal, whole protoplasts and vacuoles isolated from them produced ethylene from ACC at similar rates. Freshly-prepared evacuolated protoplasts had lost the capacity to produce ethylene. Re-formation of the central vacuole in these evacuolated protoplasts occurred between 14 to 17 h of incubation in the recovery medium and was followed by the development of ethyleneforming activity. Both these processes were inhibited by cycloheximide, indicating a requirement for new protein synthesis. Light stimulated the conversion of ACC to ethylene in both the regenerating, whole protoplasts and the evacuolated protoplasts that had re-formed the central vacuole.

Recent advances in the protoplast biology of flower crops

Abstract Gene transfer offers important opportunities to develop new plant forms, with characteristics that may not be transmitted easily by normal sexual hybridization within a species, or may not be possible in distantly related species. One of the major obstacles to applying genetic engineering technology to practical plant improvement is the use of suitable procedures for the regeneration of genetically engineered cells into whole plants. This paper reviews the current state of the art in protoplast regeneration of flower crops. The literature on protoplast culture, regeneration into whole plasts, basic biology and genetic manipulation was reviewed from 1979 to 1986. Most of the work concentrated on the Petunia and Nicotiana genera. To date, 47 different flower species have been regenerated from protoplasts to whole plants.

Determination of ploidy level in Mitchell Petunias

Abstract Mitchell Petunia plants were analyzed for their level of ploidy by: (1) counting the number of chromosomes in root tips; (2) counting the number of chloroplasts per guard cell pair; and (3) using fluorescence microscopy to measure the amount of DNA stained with Hoechst 33258. The majority of plants propagated through tissue culture were chimeric rather than either completely diploid or haploid, and we have shown that one cannot distinguish between haploids and chimeric haploid/diploid plants based on chloroplast counts. One must use either chromosome counts or microfluorimetry to distinguish between these.

Chromosomes from Protoplasts -- Isolation, Fractionation, and Uptake

We have developed methods for the isolation of condensed mitotic and meiotic chromosomes from plant protoplasts. The chromosomes can be isolated from a variety of tissues: meiocytes, root tips, cell cultures, and from a variety of species: tobacco, tomato, lily, daylily, onion, and peas. The limiting steps are the ability to obtain synchronously dividing cells and conversion of those cells into protoplasts. We have also been able to demonstrate uptake of isolated chromosomes by recipient protoplasts with fluorescence microscopy at a frequency of 1%. We could partially fractionate chromosomes by size, but we also, unfortunately, were able to show that the in vitro size did not correspond well to the relative in vivo size, so the fractionation method was not noticeably useful. We have not obtained an actual genetic transformation as yet.