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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]

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]

Solanum commersonii and Solanum tuberosum are closely related plant species that differ in their abilities to cold acclimate; whereas S. commersonii increases in freezing tolerance in response to low temperature, S. tuberosum does not. In Arabidopsis thaliana, cold-regulated genes have been shown to contribute to freezing tolerance, including those that comprise the CBF regulon, genes that are controlled by the CBF transcription factors. The low temperature transcriptomes and CBF regulons of S. commersonii and S. tuberosum were therefore compared to determine whether there might be differences that contribute to their differences in ability to cold acclimate. The results indicated that both plants alter gene expression in response to low temperature to similar degrees with similar kinetics and that both plants have CBF regulons composed of hundreds of genes. However, there were considerable differences in the sets of genes that comprised the low temperature transcriptomes and CBF regulons of the two species. Thus differences in cold regulatory programmes may contribute to the differences in freezing tolerance of these two species. However, 53 groups of putative orthologous genes that are cold-regulated in S. commersonii, S. tuberosum, and A. thaliana were identified. Given that the evolutionary distance between the two Solanum species and A. thaliana is 112–156 million years, it seems likely that these conserved cold-regulated genes—many of which encode transcription factors and proteins of unknown function—have fundamental roles in plant growth and development at low temperature.

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.

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants were transformed with a gene for choline oxidase (codA) from Arthrobacter globiformis. The gene product (CODA) was targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). These three transgenic plant types accumulated different amounts and proportions of glycinebetaine (GB) in their chloroplasts and cytosol. Targeting CODA to either the cytosol or both compartments simultaneously increased total GB content by five- to sixfold over that measured from the chloroplast targeted lines. Accumulation of GB in codA transgenic plants was tissue dependent, with the highest levels being recorded in reproductive organs. Despite accumulating, the lowest amounts of GB, Chl-codA plants exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses. This suggests that chloroplastic GB is more effective than cytosolic GB in protecting plant cells against chilling, high salt and oxidative stresses. Chloroplastic GB levels were positively correlated with the degree of oxidative stress tolerance conferred, whereas cytosolic GB showed no such a correlation. Thus, an increase in total GB content does not necessarily lead to enhanced stress tolerance, but additional accumulation of chloroplastic GB is likely to further raise the level of stress tolerance beyond what we have observed.

The tolerance of various species of plant to abiotic stress has been enhanced by genetic engineering with certain genes. However, the use of such transgenes is often associated with negative effects on growth and productivity under non-stress conditions. The codA gene from Arthrobacter globiformis is of particular interest with respect to the engineering of desirable productive traits in crop plants. The expression of this gene in tomato plants resulted in significantly enlarged flowers and fruits under non-stress conditions. The enlargement of flowers and fruits was associated with high levels of glycinebetaine that accumulated in reproductive organs, such as flower buds and fruits. The enlargement of flowers was related to an increase in the size and number of cells, and reflected the pleiotropic effect of the codA transgene on the expression of genes involved in the regulation of cell division.

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.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

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).

A protocol was developed for production of transgenic iris plants ( Iris germanica L. 'Skating Party') from regenerable suspension cultures via Agrobacterium-mediated transformation. We tested a series of selection agents, and identified hygromycin and geneticin as the most suitable for selecting transformed iris cells. Suspension cultures of iris were cocultured for 3 days with A. tumefaciens LBA 4404(pTOK233) carrying an intron-interrupted uidA (GUS) gene encoding β-glucuronidase, and hpt (hygromycin) and nptII (geneticin) selectable marker genes. Hygromycin- or geneticin-resistant calli having GUS enzyme activity were identified and used to induce plant regeneration. More than 300 morphologically normal transgenic iris plants were obtained in ≈6 months. About 80% of the transformants were GUS-positive and NPTII- positive (paromomycin-resistant). Integration of transgenes into the nuclear genome of iris plants was confirmed by Southern blot analysis. We have, therefore, developed an efficient A. tumefaciens-mediated transformation system for Iris germanica, which will allow future improvement of this horticulturally important ornamental monocot via genetic engineering. mation). Microprojectile bombardment and direct gene transfer to protoplasts are used commonly to transform a variety of monocoty- ledonous plants (Vain et al., 1995). However, stable (integrative) transformation of only two horticulturally important ornamental monocots, Dendrobium Swartz orchid (Kuehnle and Sugii, 1992) and Gladiolus L. (Kamo et al., 1995), by microprojectile bombard- ment have been reported. Agrobacterium-mediated transformation has significant advan- tages over other approaches such as integrating a few copies of T- DNA with defined border sequences and minimal rearrangement in the plant genome, preferential integration into transcriptionally active regions of the chromosome, high quality and fertility of resultant transgenic plants, and easy manipulation (Komari et al., 1998; Tingay et al., 1997). Methods for transforming dicotyledonous species with Agro- bacterium are well established. In contrast, until recently monocoty- ledons were considered beyond the range of A. tumefaciens transfor- mation methods. Various attempts to infect monocots with Agro- bacterium were made in the 1970s and 1980s, but no conclusive evidence of integrative transformation was reported (Conner and Dommisse, 1992; Smith and Hood, 1995). Successful A. tumefa- ciens-mediated transformation, however, is now possible in several agronomically important monocots including corn (Zea mays L.), wheat (Triticum aestivum L.), rice (Oryza sativa L.), barley ( Hor- deum vulgare L.), and sugar cane ( Saccharum spp. L.) (Arencibia et al., 1998; Cheng et al., 1997; Hiei et al., 1994; Ishida et al., 1996; Tingay et al., 1997). The utility of A. tumefaciens for stable (integrative) transformation of ornamental monocots was demon- strated only in Anthurium scherzerianum Schott 'Rudolph' and 'UH1060' (Chen and Kuehnle, 1996). We report here on the development of an efficient A. tumefaciens-mediated transforma- tion method for Iris.

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.

M.T. Pino, A. Avila, A. Molina, Z. Jeknic, and T.H.H. Chen. 2013. Enhanced in vitro drought tolerance of Solanum tuberosum and Solanum commersonii plants overexpressing the ScCBF1 gene. Cien. Inv. Agr. 40(1):171-184. Cultivated potato crops are sensitive to drought stress, reducing yield and tuber quality when the soil water potential drops to -0.3 MPa. However, drought not only affects plant growth and physiological activity, but this stress also induces biochemical and molecular changes in the cellular gene expression profile, triggering genes that play a direct role in plant protection and gene regulation. The genes involved in regulation include C-repeat Binding Factors/Dehydration responsive element binding (CBF/DREB) transcription factors, which increase cold, drought and salt tolerance in different plant species. The aim of this research was to evaluate whether the overexpression of the ScCBF1 gene from Solanum commersonii enhances drought stress tolerance in transgenic Solanum tuberosum and S. commersonii plantlets grown in vitro and induces drought adaptation mechanisms, such as osmoprotectors and genes for osmotic adjustment and membrane stability. Drought conditions were simulated through the addition of polyethylene glycol (PEG4000) to hormone-free MS medium. The vegetative growth, root development, proline content, and ScCBF1, Δ1-pyrroline-5-carboxylate synthetase (P5CS) and Dehydrins like genes (DNH10) expression were evaluated. The constitutive in vitro overexpression of ScCBF1 in both potato species showed better overall plantlet growth and root development under drought stress and higher proline levels in the stems and leaves. A significant increase in DNH10 expression was also associated with drought stress. In summary, the expression of ScCBF1 in potatoes induces responses associated with drought adaptation mechanisms, resulting in better overall plant growth.

C-Repeat Binding Factors (CBFs) are DNA-binding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar ‘Golden Promise’. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed ‘Golden Promise’. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of ‘Golden Promise’ and paralleled the freezing tolerance of the winter hardy barley ‘Dicktoo’. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in ‘Golden Promise’. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in ‘Golden Promise’ both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures.

Irises are perennial plants widely used as ornamental garden plants or cut flowers. Some species accumulate secondary metabolites, making them highly valuable to the pharmaceutical and perfume industries. Micropropagation of irises has successfully been accomplished by culturing zygotic embryos, different flower parts, and leaf base tissues as starting explants. Plantlets are regenerated via somatic embryogenesis, organogenesis, or both processes at the same time depending on media composition and plant species. A large number of uniform plants are produced by somatic embryogenesis, however, some species have decreased morphogenetic potential overtime. Shoot cultures obtained by organogenesis can be multiplied for many years. Somatic embryogenic tissue can be reestablished from leaf bases of in vitro-grown shoots. The highest number of plants can be obtained by cell suspension cultures. This chapter describes effective in vitro plant regeneration protocols for Iris species from different types of explants by somatic embryogenesis and/or organogenesis suitable for the mass propagation of ornamental and pharmaceutical irises.

Irises are perennial plants widely used as ornamental garden plants or cut flowers. Some species accumulate secondary metabolites, making them highly valuable to the pharmaceutical and perfume industries. Micropropagation of irises has successfully been accomplished by culturing zygotic embryos, different flower parts, and leaf base tissues as starting explants. Plantlets are regenerated via somatic embryogenesis, organogenesis, or both processes at the same time depending on media composition and plant species. A large number of uniform plants are produced by somatic embryogenesis, however, some species have decreased morphogenetic potential overtime. Shoot cultures obtained by organogenesis can be multiplied for many years. Somatic embryogenic tissue can be reestablished from leaf bases of in vitro-grown shoots. The highest number of plants can be obtained by cell suspension cultures. This chapter describes effective in vitro plant regeneration protocols for Iris species from different types of explants by somatic embryogenesis and/or organogenesis suitable for the mass propagation of ornamental and pharmaceutical irises.

†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.

Solanum tuberosum is a frost-sensitive species incapable of cold acclimation. A brief exposure to frost can significantly reduce its yields, while hard frosts can completely destroy entire crops. Thus, gains in freezing tolerance of even a few degrees would be of considerable benefit relative to frost damage. The S. tuberosum cv. Umatilla was transformed with three Arabidopsis CBF genes (AtCBF1-3) driven by either a constitutive CaMV35S or a stress-inducible Arabidopsis rd29A promoter. AtCBF1 and AtCBF3 over-expression via the 35S promoter increased freezing tolerance about 2 degrees C, whereas AtCBF2 over-expression failed to increase freezing tolerance. Transgenic plants of AtCBF1 and AtCBF3 driven by the rd29A promoter reached the same level of freezing tolerance as the 35S versions within a few hours of exposure to low but non-freezing temperatures. Constitutive expression of AtCBF genes was associated with negative phenotypes, including smaller leaves, stunted plants, delayed flowering, and reduction or lack of tuber production. While imparting the same degree of freezing tolerance, control of AtCBF expression via the stress-inducible promoter ameliorated these negative phenotypic effects and restored tuber production to levels similar to wild-type plants. These results suggest that use of a stress-inducible promoter to direct CBF transgene expression can yield significant gains in freezing tolerance without negatively impacting agronomically important traits in potato.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

Low-temperature stress, including freezing and chilling, is one of the primary factors that limits crop production around the world. A severe winter or unseasonable cold spell in a major agricultural area can reduce yield, delay harvest, lower the quality of the products, and/or cause crop failure. Low-temperature stress is not limited to particular geographic regions; producers throughout the world have suffered significant economic losses during the past decade. These losses can significantly influence the economic well-being of rural communities, and affect product availability and consumer prices. Stress can also have a great impact on the stability of world food supplies, and may subsequently influence commodity prices and the international trade balance.

In poplar (Populus deltoides Bartr.ex Marsh), the development of bud dormancy is initiated by short-day (SD) photoperiods. The degree of bud dormancy, expressed as days to budbreak, increased from ≈10 days for plants grown under long-days to >200 days after 10 weeks of SD exposure. We investigated quantitative and qualitative changes in protein fractions extracted from terminal buds, lateral buds, bark, and leaves of poplar plants during the induction of bud dormancy by 2-D PAGE. While total protein contents(as milligrams per gram fresh weight) in leaves, terminal, and lateral buds did not change significantly during SD treatment, bark protein content increased about five-fold in 10 weeks. The results of 2-D PAGE analysis indicated that there was a significant change in protein profiles in terminal and lateral buds, leaves, and bark. The results suggested that SD treatment in poplar plants causes substantial changes in protein profiles during the induction of bud dormancy.

Iz listova zaraženih primjeraka korovne biljke Erigeron canadensis L. koji su rasli na nekoliko lokaliteta u Sarajevu izoliran je virus iz skupine tombusvirusi. Identifikacija virusa izvršena je na osnovi reakcije pokusnih biljaka, analize ultratankih presjeka kroz zaraženo tkivo, morfologije i veličine virusnih čestica, te na osnovi seroloških reakcija metodom dvostruke imunodifuzije u agarskom gelu i čvrsto fazne imunoelektronske mikroskopije (SPIEM). U serološkim pokusima upotrijebljen je imuni serum protiv špinatskog soja virusa grmolike kržljavosti rajčice. Ovim je radom vrsta E. canadensis prvi put zabilježena kao prirodni domaćin jednog tombusvirusa., Many samples of the species Erigeron canadensis L. with marked symptoms of viral disease were found in many locations in Sarajevo area, Yugoslavia. A virus belonging to the group of tombusviruses was isolated from infected plants. The identification of the virus is based on reactions of diagnostic herbaceous species, characteristic cytopathogenic changes, morphology and size of viral particles, serological test of double diffusion in agar gel, and immune electron microscopy by »trapping« method (SPIEM). In serological test the serum against spinach strain of tomato bushy stunt virus was used. This experiment, has shown for the first time that the species Enigeron canadensis is a natural host plant for a tombusvirus.