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Genetic modulation of the carotenogenesis in I. germanica `Fire Bride' by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors. Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, `Fire Bride', with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica `Fire Bride' and produce new flower traits. Cooley's Gardens Inc., Silverton, OR {[}ARF3711]; Ministry of Education, Science and Technological Development, Republic of Serbia {[}TR31019] more...

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two kappa-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene beta-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native kappa-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of kappa-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species. Cooleys Gardens Inc., Silverton, OR [ARF3711]; National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH) [P30ES000210, S10RR027878]; Ministry of Education and Science, Republic of Serbia [TR31019] more...

We studied the effect of ectopic AtCBF over-expression on physiological alterations that occur during cold exposure in frost-sensitive Solanum tuberosum and frost-tolerant Solanum commersonii. Relative to wild-type plants, ectopic AtCBF1 over-expression induced expression of COR genes without a cold stimulus in both species, and imparted a significant freezing tolerance gain in both species: 2 degrees C in S. tuberosum and up to 4 degrees C in S. commersonii. Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of wild-type S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild type, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest an endogenous CBFpathway is involved in many of the structural, biochemical and physiological alterations associated with cold acclimation in these Solanum species. more...

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants are chilling sensitive, and do not naturally accumulate glycinebetaine (GB), a metabolite that functions as a stress protectant. We reported previously that exogenous GB application enhanced chilling tolerance in tomato. To understand its protective role better, we have further evaluated various parameters associated with improved tolerance. Although its effect was most pronounced in younger plants, this benefit was diminished 1 week after GB application. When administered by foliar spray, GB was readily taken up and translocated to various organs, with the highest levels being measured in meristematic tissues, including the shoot apices and flower buds. In leaves, the majority of endogenous GB was found in the cytosol; only 0.6-22.0% of the total leaf GB was localized in chloroplasts. Immediately after GB application, levels of H(2)O(2), catalase activity and expression of the catalase gene (CAT1) were all higher in GB-treated than in control plants. One day after exposure to chilling stress, the treated plants had significantly greater catalase activity and CAT1 expression, although their H(2)O(2) levels remained unchanged. During the following 2 d of this chilling treatment, GB-treated plants maintained lower H(2)O(2) levels but had higher catalase activity than the controls. These results suggest that, in addition to protecting macromolecules and membranes directly, GB-enhanced chilling tolerance may involve the induction of H(2)O(2)-mediated antioxidant mechanisms, e.g. enhanced catalase expression and catalase activity. more...

To improve the efficiency of iris plant regeneration, we tested the influence of several combinations of Kin and NAA in culture media on the induction of morphogenesis and the subsequent development of plantlets. The highest rates of regeneration (67%) were consistently observed in induction media containing 0.5 μm NAA and either 2.5 or 12.5 μm Kin. Developing medium containing 1.25 μm BA was optimal for high regeneration rates and a high percentage of plantlets simultaneously developing shoots and roots. Rooted plantlets were easily acclimatized and transplanted to various soil mixtures, then grown in the greenhouse. Under optimal conditions as many as 8000 plantlets could be regenerated from 1 g of cells in ≈4 months. Chemical names used: kinetin (Kin); 1-naphthaleneacetic acid (NAA); N6-benzyladenine (BA). more...

A protocol was developed for efficient plant regeneration of Iris germanica L. 'Skating Party' from suspension cultures. Suspension cultures were maintained in Murashige and Skoog (MS) basal medium (pH 5.9) supplemented with 290 mg·L -1 proline, 50 g·L -1 sucrose, 5.0 μM 2,4-D, and 0.5 μM Kin. Suspension-cultured cells were transferred to a shoot induction medium (MS basal medium supplemented with 10 mg·L -1 pantothenic acid, 4.5 mg·L -1 nicotinic acid, 1.9 mg·L -1 thiamine, 250 mg·L -1 casein hydrolysate, 250 mg·L -1 proline, 50 g·L -1 sucrose, 2.0 g·L -1 Phytagel, 0.5 μM NAA, and 12.5 μM Kin). Cell clusters that proliferated on this medium differentiated and developed shoots and plant- lets in about 5 weeks. Regeneration apparently occurred via both somatic embryogenesis and shoot organogenesis. A series of experiments was conducted to optimize conditions during suspension culture to maximize subsequent plant regeneration. Parameters in- cluded 2,4-D and Kin concentrations, the subculture interval, and the size of cell clusters. The highest regeneration rate was achieved with cell clusters ≤280 μm in diameter, derived from suspension cultures grown for 6 weeks without subculturing in liquid medium containing 5 μM 2,4-D and 0.5 μM Kin. Up to 4000 plantlets with normal vegetative growth and morphology could be generated from 1 g of suspension-cultured cells in about 3-4 months. Chemical names used: 2,4-dichlorophenoxyacetic acid (2,4-D); kinetin (Kin); 1- naphthaleneacetic acid (NAA). Iris germanica is one of the horticulturally most important tall bearded irises in the genus. Hundreds of valuable cultivars from this spe- cies have been developed and cultivated com- mercially as perennial ornamental plants. Tra- ditionally, rhizomatous iris plants are propa- gated by splitting rhizomes, with a maximum annual yield of 10 plants/rhizome (Jehan et al., 1994). This practice is inefficient and slow, especially for propagating new cultivars for commercial use. Propagation by seed is im- practical because of low germination rates and the allogamous nature of iris. Therefore, a more efficient propagation method is needed. Also, strong consumer demand increases the challenge of developing new cultivars with novel flower characteristics. Introducing de- sirable traits by genetic transformation would offer an attractive means for iris cultivar im- provement. more...

†Background and Aims Lateral root initiation is an essential and continuous process in the formation of root systems; therefore, its quantitative analysis is indispensable. In this study a new measure of lateral root initiation is proposed and analysed, namely the lateral root initiation index (ILRI), which defines how many lateral roots and/or primordia are formed along a parent-root portion corresponding to 100 cortical cells in a file. †Methods For data collection, a commonly used root clearing procedure was employed, and a new simple root clearing procedure is also proposed. The ILRI was determined as 100dl, where d is the density of lateral root initiation events (number mm 21 ) and l is the average fully elongated cortical cell length (mm). †Key Results Analyses of different Arabidopsis thaliana genotypes and of a crop plant, tomato (Solanum lycopersicum), showed that ILRI is a more precise parameter than others commonly used as it normalizes root growth for variations in cell length. Lateral root primordium density varied in the A. thaliana accessions Col, Ler, Ws, and C24; however, in all accessions except Ws, ILRI was similar under the same growth conditions. The nitrogen/carbon ratio in the growth medium did not change the lateral root primordium density but did affect ILRI. The ILRI was also modified in a number of auxin-related mutants, revealing new root branching phenotypes in some of these mutants. The rate of lateral root initiation increased with Arabidopsis seedling age; however, ILRI was not changed in plants between 8 and 14 d post-germination. †Conclusions The ILRI allows for a more precise comparison of lateral root initiation under different growth conditions, treatments, genotypes and plant species than other comparable methods. more...