Flavonoids, including anthocyanins are ubiquitous compounds constituting about 5-10% of the known secondary metabolites imparting vivid floral, seed and foliage colors in plants ranging from bryophytes to angiosperms. Plants are specialized in synthesizing and accumulating specific combinations of flavonoids out of a pool of about 5000 known flavonoids implying their adaptive functions. The flavonoid pathway has been well characterized in a few select plants. The genetic and molecular analysis revealed that the pathway is governed by a number of loci dispersed across the plant genome and regulated by distinct regulatory gene families in a temporal and spatial manner. With the rapid growth in molecular and biochemical characterization of the genes and their products it has now become possible to precisely elucidate the role of flavonoids in plant survival. Flavonoids have been implicated in diverse functions such as UV-B protection, signal molecules in plant-microbe symbiotic associations, plant defense response, cold stress response, modulators of hormone response and pollen fertility. In addition, the role of flavonoids as very powerful dietary anti-oxidant supplements in human nutrition is increasingly demonstrated. The review highlights certain structural and functional aspects of flavonoids particularly their role in stress response. Further, recent advances in application of biotechnology tools to manipulate flavonoid pathway in different plants has been described. Flavonoid genes as benign and visible reporters of plant origin in transformation experiments appears to be promising. Studies on transgenic plants carrying genetically engineered flavonoid genes leading to the accumulation of flavonoids by sense over-expression or decrease or elimination by anti-sense suppression respectively, have been used to manipulate plants defense response against bacterial and fungal diseases. Flavonoid biotechnology has become a powerful tool to manipulate flower color in horticulture industry. This review critically evaluates various functions of flavonoids and describes specific instances and strategies of biotechnological manipulation to improve plant performance and value addition.
Direct and callus-mediated shoot regeneration following co-cultivation with Agrobacterium tumefaciens was obtained in two Indian cultivars of safflower (Carthamus tinctorius L.), A-1 and A-300. The procedure yielded 23 and 34 transformation events per 100 co-cultivated explants with direct and callus-mediated shoot recovery, respectively. The use of uid A gene in pKIWI105 that lacks a bacterial ribosome binding site precluded uid A expression in residual Agrobacterium cells. High levels of GUS activities were detected in selected putative transgenic calli and in shoot regenerants by histochemical assay. Western blot analyses using GUS antiserum and the NPT II expression assays confirmed the expression of marker genes in the putative transformants. Transgene integration was examined by PCR and dot blot hybridization of the transformants. Compared to controls, the efficiency of regeneration was markedly decreased subsequent to co-cultivation. Extended periods of callusmediated regeneration led to hyperhydricity and vitrification of the shoots. Shoots regenerated from explants directly had, however, a normal appearance. The rooting response of regenerated shoots was poor and remains a continuing obstacle for safflower plant regeneration and transformation.
Conditions for rapid propagation of high-quality garlic seed-bulb derived from meristem-tips were investigated. Meristem-tips of garlic ‘Howaito-roppen’ were excised with or without a leaf primordium, and propagated to produce in vitro bulblets. Cloves were formed through one year of cultivation of the bulblets. Western blots for the obtained garlic plants indicated that meristem-tips without a leaf primordium to be a more suitable material to obtain healthy plants. Evaluation of clove formation could be performed on the column diameter at mid May. The clove formation depended on the size of the plant at the time of formation of clove initials. Plants without symptoms of virus were obtained irrespective of the size of the meristem-tips excised. However, some of the plants with no mosaic symptoms reacted with antiserum of garlic viruses, and portions of these plants increased when meristem-tips were cultured with leaf primordium. The resultant bulbs were cultivated in farmer's fields to investigate re-infected viruses. Sixty percent of the plants exhibited mosaic symptoms after 5 years of cultivation, the major viruses being LYSV and GMbMV. Re-infection was prevented by culturing bulbs in an isolated field.
A reproducible system for production of transgenic plants via Agrobacterium mediated transformation of pigeonpea (Cajanus cajan L.) was developed. Shoot apices and cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain LBA4404. The strain harbours a binary vector carrying the reporter gene (β-glucuronidase (uidA) and the marker gene neomycin phosphotransferase (nptII). Cocultivated explants were cultured on shoot regeneration medium with 2mgl-1 BAP and kanamycin (50μgml-1 for selection (MS1). Approximately 45-62% of the explants produced putatively transformed shoots on the selection medium. Multiple shoots were repeatedly selected on 0.5mgl-1 BAP and 25μgml-1 kanamycin medium (MS2) The elongated shoots were subsequently rooted on a medium supplemented with 25μgml-1 kanamycin sulfate (MS3). The transgenic plants were later established in pots. Although transformation was achieved with both cotyledonary node and shoot apices, cotyledonary nodes responded better with 62% of the explants producing GUS positive shoots after selection on MS2 medium. The presence of uidA and nptII genes in the transgenic plants was verified by PCR analysis. Integration of T-DNA into the genome of transgenic plants was further confirmed by Southern blot analysis.
Allergenic proteins of molecular mass 22kDa, 36kDa, 39-40kDa and 70-72kDa were identified from water-soluble fractions of common buckwheat cv. “Miyazaki-zairai”. The identification was based on the reactivity of the protein with IgE antibodies from patients' allergic to buckwheat. The allergenic proteins fall within the β and γ subunits of the globulin fraction indicating that the allergenic reactions are contributed by the major storage protein in buckwheat. The 22kDa protein was the most consistent with IgE activity. N-terminal amino acid sequence of 22kDa protein showed maximum homology with the seed globulins from both dicot and monocot species. Based on the N-terminal amino acid sequence, two degenerate primers were selected to amplify the cDNA encoding the major allergenic protein. A single PCR product obtained was subsequently sequenced. The deduced amino acid sequence was identical in its N-terminal end. Homology search revealed 94 and 93% homology to two buckwheat legumin like proteins.
In vitro chromosome doubling by the use of the variation induced by zeatin riboside was applied to generate tetraploid individuals to introgress potato tuber moth (PTM) resistance from diploids to tetraploids. This approach is especially important when the expression of desirable traits is strongly influenced by cytoplasmic factors. The levels of PTM resistance and yield performance was evaluated in chromosome-doubled regenerates and their corresponding original diploids. The levels of PTM resistance were maintained in the derivatives. Test of yield performance in two years indicated chromosome-doubled regenerates generally had comparable yield with their diploid original potato clones. These results demonstrated that in vitro chromosome doubling could be an effective method to convert valuable 2x potato germplasm into the 4x level without the considerable loss of PTM resistance nor productivity.
Phytosulfokine-α (PSK-α) is a novel plant peptidyl growth factor that was originally isolated from the conditioned medium of cultures of asparagus mesophyll cells and was shown to stimulate cell division. To date, however, there have been no reports on the effects of PSK-α on morphogenesis in higher plants. Therefore, in this study, we examined the effects of PSK-α on formation of adventitious buds from calli of Antirrhinum majus. The formation of adventitious buds was induced on hypocotyl-derived callus that had formed on solid Murashige and Skoog's (MS) medium containing 1.2μM naphthoxyacetic acid. Addition of PSK-α in this medium at 10-8, 10-7 and 10-6M stimulated the formation of adventitious buds. No adventitious buds developed from callus that had been cultured on MS medium containing 1.1μM 2, 4-D with or without PSK-α. However, when such callus was transferred to phytohormone-free MS medium that contained PSK-α, as many as 10% of calli formed adventitious buds. The results indicate that PSK-α could stimulate organogenesis in higher plant.
Effects of the five macro-components and sucrose in half strength MS (1/2MS) agar medium on red-color pigmentation were studied in the plant bodies of Dionaea muscipula and Drosera spathulata generated from multiple shoots in vitro. In 1/2MS agar medium modified with 10.31mM NH4NO3 and 9.40mM KNO3 and supplemented with 0.75 or 0% sucrose the subcultured plants continuously proliferated by multiple shoots and generated large, green-colored plants, while with dilution of those nitrogen components and increase of sucrose to 1.5% the red-color anthocyanin pigmentation spread from the glands or glandular hairs to the entire leaves and the plant sizes and dry weight decreased in inverse proportion to the depth of red color. The anthocyanin pigments of Dionaea muscipula consisted of delphinidin 3-O-glucoside which was new to the species and cyanidin 3-O-glucoside (chrysanthemin), and those of Drosera spathulata, studied here for the first time, consisted of cyanidin 3, 5-di-O-glucoside (cyanin), cyanidin 3-O-galactoside (idaein), cyanidin 3-O-glucoside, pelargonidin 3-O-galactoside, and pelargonidin 3-O-glucoside (callistephin).
Leaf explants of four species of licorice (Glycyrrhiza glabra L., G. uralensis F., G. echinata L., and G. squamulosa F.) were cultured on Murashige and Skoog (MS) basal medium. G. glabra L. and G. uralensis F. formed adventitious roots from calli on the medium containing NAA (1mgl-1). Both calli induced from G. echinata L. and G. squamulosa F. formed somatic embryos on the medium containing NAA (1mgl-1) and BAP (1mgl-1), or 2, 4-D (0.5mgl-1) alone. Plantlets regenerated from the somatic embryos were transplanted to soil.
Long-term storage of in vitro-grown wasabi (Wasabia japonica MATSUMURA) meristems at low temperature using dimethylsulfoxide (DMSO) were investigated. Meristems were plated on solidified 1/2MS medium supplemented with 3% sucrose and 1% DMSO for a day, and then they were successfully stored at -5°C for 24 months. After the storage, meristems were recultured on solidified 1/2MS medium supplemented with 3% sucrose and 0.1mgl-1 6-benzyladenine (BA). The survival rate was about 60%. Although many meristems appeared water-soaked after storage, most recovered to normal shoots.
Long-term maintenance of plant cells in tissue culture is known to induce somaclonal variation in regenerated plants. Here we examined somaclonal variation in regenerated plants of Easter lily (Lilium longiflorum Thunb.) using cytological and molecular analyses. Forty nine 10-mer and 12-mer arbitrary primers were used to amplify DNAs extracted from sixty plants regenerated after more than two years of callus culture originally derived from bulb tissues. Sixteen primers were found to produce 34 polymorphic bands in the regenerated plants, including both loss of parental bands and appearance of novel, non-parental bands. The presence of polymorphic fragments among the regenerants indicates that genomic alterations occurred during the long-term culture of the cells. Although no variation was observed from karyotype analysis, the present study revealed the high frequency of the occurrence of somaclonal mutations at the DNA level during the callus culture of Easter lily.