). The Genbank accession number for Citrus sinensis 1,6RhaT is DQ119035. SUMMARYDomestication and breeding of citrus species/varieties for flavor and other characteristics, based on the ancestral species pummelo, mandarin and citron, has been an ongoing process for thousands of years. Bitterness, a desirable flavor characteristic in the fruit of some citrus species (pummelo and grapefruit) and undesirable in others (oranges and mandarins), has been under positive or negative selection during the breeding process of new species/varieties. Bitterness in citrus fruit is determined by the composition of branched-chain flavanone glycosides, the predominant flavonoids in citrus. The flavor-determining biosynthetic step is catalyzed by two branch-forming rhamnosyltransferases that utilize flavanone-7-O-glucose as substrate. The 1,2-rhamnosytransferase (encoded by Cm1,2RhaT) leads to the bitter flavanone-7-O-neohesperidosides whereas the 1,6-rhamnosytransferase leads to the tastelessflavanone-7-O-rutinosides. Here, we describe the functional characterization of Cs1,6RhaT, a 1,6-rhamnosyltransferase-encoding gene directing biosynthesis of the tasteless flavanone rutinosides common to the non-bitter citrus species. Cs1,6RhaT was found to be a substrate-promiscuous enzyme catalyzing branched-chain rhamnosylation of flavonoids glucosylated at positions 3 or 7. In vivo substrates include flavanones, flavones, flavonols and anthocyanins. Cs1,6RhaT enzyme levels were shown to peak in young fruit and leaves, and gradually subside during development. Phylogenetic analysis of Cm1,2RhaT and Cs1,6RhaT demonstrated that they both belong to the branch-forming glycosyltransferase cluster, but are distantly related and probably originated separately before speciation of the citrus genome. Genomic data from citrus, supported by a study of Cs1,6RhaT protein levels in various citrus species, suggest that inheritance, expression levels and mutations of branchforming rhamnosyltransferases underlie the development of bitter or non-bitter species/varieties under domestication.
A protocol for plantlet regeneration through shoot formation was developed for the neotropical shrub Brunfelsia calycina. This shrub is unique in its change in flower color from dark purple to white. Explants from young and mature leaves were incubated on MS medium (pH 5.7, 30 g/l sucrose, 7.5 g/l agar) with various combinations of Indole-3-acetic acid (IAA) and 6-Benzyladenine (BA) under a 16 h photoperiod at a constant temperature of 25°C. Shoot emergence was best at 4.44 lM BA and 2.85 lM IAA for young leaf explants, and at 8.88 lM BA, 2.85 lM IAA for mature leaf explants. When shoots were transferred to MS medium supplemented with 1.23-2.46 lM indole butrytic acid (IBA), they developed roots. KeywordsRegeneration Á Solanaceae Á Brunfelsia Abbreviations Aux Auxin BA 6-Benzyladenine Ck Cytokinin IAA Indole-3-acetic acid IBA Indole-3-butyric acid MS Murashige and Skoog (1962) NAA 2-Naphthaleneacetic acid TDZ Thidiazuron (N-phenyl N 0 1,2,3-thidiazol-5-yl urea) Brunfelsia (Solanaceae) is a genus of about 40 species of shrubs and small trees native to Brazil. The typical habitat for these plants is light woodland and thickets. Several Brunfelsia species contain medicinal and toxic alkaloids. For example, B. grandiflora is the source of the most important native remedies employed against rheumatism, arthritis and snake bites in the upper Amazon region (Plowman 1977). Other Brunfelsia species, among them B. calycina, have been cultivated as ornamentals. B. calycina has become a popular garden and pot plant due to its large blue flowers and pleasant fragrance (Heide 1963). A striking characteristic of B. calycina flowers is their rapid color change from dark purple on the day of opening to white within only 3 days, resulting in bushes with both purple and white flowers. Because of this rapid color change, their common name is Yesterday-Today-Tomorrow (Brenzel 2001; Halevy 1985; Heide 1963). We have shown that the color change in B. calycina flowers is due to degradation of anthocyanins and that this process is dependent on the synthesis of novel proteins (Vaknin et al. 2005). There is very little information about anthocyanin degradation in plants, thus B. calycina can serve as a model plant for understanding this process. A first step in testing the involvement of candidate genes in the anthocyanindegradation process is the development of a regeneration protocol for B. calycina. Here we present a method for regenerating B. calycina plantlets from leaf explants. To the best of our knowledge, this is the first published regeneration protocol for Brunfelsia.We tested the regeneration potential of several plant organs on a variety of media. Flower buds, axial buds, nonwoody green stems, young leaves that are still expanding (up to five leaves from the apical meristem) and fully expanded mature leaves were cut from B. calycina plants grown in pots in a glass greenhouse under controlled Raya Liberman and Liat Shahar have equally contributed to this work.
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