A simple and reliable regeneration system from leaf bases of barley (Hordeum vulgare L.) has been developed. The in vitro regeneration frequencies of seven commercial barley genotypes were compared using segments from the first leaves of 5-d-old seedlings. The regeneration frequency ranged from 31.56 to 72.22 % among the barley genotypes. Murashige and Skoog medium supplemented with 6-benzyladenine (0.5 mg dm^-3) and kinetin (0.5 mg dm^-3) was optimum for the regeneration. Longitudinal cut of the segments or the removal of coleoptiles further increased plantlet regeneration frequency.

To characterize the low molecular mass glutenin subunit gene 177-21 (AY994364) in wheat (Triticum aestivum L. cv. Jinan 177), we developed a specific PCR primer set to decide its locus with nullisomic-tetrasomic lines of Chinese spring wheat. The result showed that it was assigned to Glu-D3. The DNA fragment of 177-21 was then subcloned into the pGEX-4T-1 expression vector and expressed in E. coli with isopropyl-1-thio-β-D-galactoside induction. The result indicated that this gene encodes about 30 kD polypeptide and deduced amino acid sequence consists of eight cysteine residues. Of the eight, six may be related with the formation of intra-molecular disulfide bonds, the last two with the formation of inter-molecular disulfide bonds, which could be a potential extender in "glutenin polymer" to have positive influence on quality of wheat flour.

A near isogenic line (NIL) of Brassica oleracea var. botrytis with resistant and susceptible lines C712 and C731, was used in this study. More than 100 differentially expressed cDNA fragments were obtained from black rot resistant cauliflower plants obtained using cDNA-amplified fragment length polymorphism (AFLP) after infection with the pathogen. Thirteen of these fragments were cloned and subjected to reverse Northern blot analysis using both infected and control cDNA pools. Two positive clones, M2 and M6, were isolated. Northern dot blot and Northern blot analyses showed that M2 was constitutively expressed, whereas M6 contained a gene that was differentially expressed during pathogen infection. Moreover, M6 cDNA fragment was also highly expressed 16-24 h after H₂O₂ treatment. Southern blots showed that M6 is a single copy gene in the cauliflower genome, and encodes a protein with 84 % homology to gene on Arabidopsis chromosome 1. The deduced M6 protein has 91 % positive homology with the Arabidopsis 2A6 protein, which regulates ethylene synthesis; 76 % homology with a 1-aminocyclopropane-1-carboxylate oxidase (ACO), the last enzyme in ethylene synthesis; and 70 % homology with an ethylene induced DNA binding factor. These results suggest that M6 gene fragment is a new H₂O₂ downstream defense related gene fragment and can be induced by Xanthomonas campestris pv. campestris and H₂O₂.

In 7-d-old seedlings of Brassica juncea chromium (VI) promoted photosystem 2 (PS 2) mediated photoreactions. The increase in PS 2 activity in the thylakoids from Cr-treated seedlings, in the presence of uncoupler (5 mM NH₄Cl), was similar to that recorded with the control thylakoids. Thus Cr enhanced PS 2 activity was not due to uncoupling of electron transport from photophosphorylation. Photon saturation kinetics revealed that the PS 2 activity of thylakoids from Cr-treated seedlings was significantly higher at almost all irradiances in comparison to that of controls. PS 2 activity of thylakoids from Cr-treated plants at 25 % of the saturating irradiance was in par with the PS 2 activity of the thylakoids from control plants at saturating irradiance. Thylakoids from both control and Cr-treated seedlings exhibited maximum PS 2 activity at pH 7.5. The PS 2 activity of thylakoids from Cr-treated plants remained high even at pH 8.0 and 8.5, demonstrating Cr enhances tolerance of PS 2 to alkaline pH.

In order to better understand the role of cold acclimation in alleviating freezing injury, two barley cultivars with different cold tolerance, i.e. a sensitive cv. Chumai 1 and a tolerant cv. Mo 103, were used. The freezing treatment increased leaf soluble protein content more in the tolerant cultivar than in the sensitive one. Cold acclimation increased H₂O₂ content of the two cultivars during freezing treatment, especially in Mo 103. Glutathione and ascorbate contents during freezing and recovery were significantly higher in cold-acclimated plants than in non-acclimated ones. Activities of peroxidase, ascorbate peroxidase and glutathione reductase were also higher in cold-acclimated plants than non-acclimated plants during freezing treatment. However, there was no significant difference between cold-acclimated plants and the control plants in catalase activity. It may be assumed that cold acclimation induced H₂O₂ production, which in turn enhanced activities of antioxidative enzymes and synthesis of antioxidants, resulting in alleviation of oxidative stress caused by freezing.

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 µM IAA, 8.87 µM BA, and 11.2 µM TRIA. The primary shoots elongated best on MS medium containing 6.66 µM BA and 2.45 µM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 µM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.

In order to evaluate effects of γ-rays on adventitious root formation and ginsenoside production, embryogenic calli induced from cotyledon explants of Panax ginseng C.A. Meyer were treated with γ-rays of 0, 10, 30, 50, 70, and 100 Gy. The highest frequency of adventitious root formation of 75 % occurred at γ-irradiation of 30 Gy, which is considered adequate dosage for selecting mutant cell lines. Five mutated adventitious roots (MAR)3-lines out of the propagation of 142 adventitious root lines treated with 30 Gy were selected based a 100-fold increase in proliferation rate compared to control adventitious roots (CAR) and content of the seven major ginsenosides (Rb₁, Rb₂, R_c, R_d, R_e, R_f, and Rg₁) was determined. In the CAR and four of the MAR3-lines (except for MAR3-109), the Rb/Rg ratio was greater than 1.0, thereby indicating altered ginsenoside composition in these root lines. The HPLC analysis of the MAR3-13 and MAR3-26 lines confirmed different ginsenoside profiles, including the three unidentified ginsenoside candidates, Gm₁, Gm₂, and Gm₃. The ginsenosides of the MAR3-13 and MAR3-26 lines showed high hydroxyl and superoxide radical scavenging activities.

Specific activities and isoform patterns of peroxidases, acid phosphatases, DNases and RNases were studied in relation to in vitro rooting of Petunia × hybrida microshoots in the presence of 4 µM indole-3-butyric acid (IBA). Specific activities of the above enzymes increased in the course of rooting. Rhizogenesis could be related with an increased specific activity of peroxidases during the initiation phase, in parallel with increased lignin content. Twelve peroxidases, six anionic (A1-A6) and six cationic (C1-C6), seven acid phosphatases (ACP1-ACP7), seven RNases (R1-R7) and four DNases (D1-D4) isoforms were detected following native PAGE. Variation in the number of the above isoforms and their quantity was observed during different stages of rooting. Particularly, A2, A3, C3, C4, C5, ACP2, R1, R2, R3, and D4 isoforms appeared after the induction phase and could be related to emergence of root primordia. Additionally, R3 and D4 could be associated with cell division and differentiation, since these are only expressed in rooted microshoots. Moreover, the higher number of roots in IBA-treated microshoots could be related to the higher expression of RNase and DNase isoforms during initiation and expression phases.

In order to study chloroplast biogenesis, we chose natural variegated Epipremnum aureum (golden pothos) and regenerated pale yellow, variegated and green plants from all three types of tissue explants. The percentage of three types of regenerated shoots from three different explants was very close. Regenerated plants have been maintained for a year and show no sign of a colour switch. By comparing their protein profiles, two major differences between pale yellow and green plants were observed at the 15 and 40 to 50 kDa proteins. Moreover, pale yellow plants had unexpected high molecular mass proteins (greater than 60 kDa). Both variegated and green plants had more chlorophyll (Chl) a than Chl b, the ratios were about 1.46 and 1.93, respectively. In contrast, the pale yellow plants not only had less total Chl, but also the reduction of Chl a was much greater than Chl b, resulting in a higher content of Chl b than Chl a. Microscopic analysis revealed that pale yellow plants contained predominantly undeveloped chloroplasts with low Chl contents, even though their mesophyll cells were similar to green and variegated plants. PCR amplification of chloroplast DNA with 14 universal chloroplast primers did not reveal any difference among these regenerated plants.

Twenty accessions of the sparsely cultivated diploid Musa cultivars Matti (AA) and Rasakadali (AB) were subjected to random amplified polymorphic DNA (RAPD) assay. A total of 14 random primers were used for the estimation of interand intracultivar variations. Out of 86 bands generated, 64 were polymorphic (74.4 % polymorphism). The cluster analysis grouped the cultivars into two major clusters: cluster I with 10 accessions of Matti and 2 of Rasakadali and cluster II comprising the remaining 8 accessions of Rasakadali. The coefficient of genetic similarity (GS) was from 0.73 to 0.99, suggesting low level of intercultivar variation. The accessions of Rasakadali with mean GS of 0.89 were genetically more diverse than those of Matti (GS = 0.93).

Molecular farming provides a powerful tool for low cost production of recombinant proteins with pharmaceutical value. The use of transgenic plants has been increasingly tested as alternative system for obtaining biologically active human lactoferrin in plants. Precise selection of plant species, transformation techniques and expression cassettes, in addition to conduction of detailed glycosylation and immunogenicity studies, serves as basis of obtaining safe recombinant human lactoferrin in high concentrations for the use of pharmacy. On the other hand, expression of antimicrobial protein lactoferrin in plants is a promising opportunity for crop quality improvement by increasing plant disease resistance.

β-1,3-Glucanase activity was detected in extracts of different tissues of healthy mature petunia flowers except the filament. The stigma was studied further as it had the highest enzyme activity and there is a paucity of information on the occurrence of this enzyme in this tissue. Specific activity of the enzyme was found to increase within the stigmatic tissue from early development until just before anthesis. Following non-denaturing polyacrylamide gel electrophoresis at pH 8.8, extracts of dehiscent stigma seem to contain three acidic isoforms of β-1,3-glucanase. Crude extracts of stigma was passed through a pachyman affinity column. A fraction of affinity-purified active β-1,3-glucanase enzyme was found to have no antifungal activity against Trichoderma viride, Phloma clematidina and Cladosporium fulvum.

The effect of in vitro cultivation of donor shoots on subsequent morphogenesis in leaf explants of quince (Cydonia oblonga Mill.) clone BA29 was investigated. Proliferating donor shoots were cultured in ventilated or closed vessels under different photosynthetic photon flux densities (PPFD; 200 and 100 µmol m^-2 s^-1) with 0, 15, 30 g dm^-3 sucrose. Shoots grown in ventilated vessels, especially with sucrose at 15 or 30 g dm^-3, were better developed with fully expanded leaves compared to those in standard closed vessels. Leaves collected from pre-treated donor shoots were used to assess regeneration capacity. Somatic embryo production was highest in leaves harvested from shoots cultured in closed vessels with 30 g dm^-3 sucrose and in ventilated vessels with 15 and 30 g dm^-3 sucrose and under high PPFD which was, in comparison with the control treatment (closed vessel, 30 g dm^-3 sucrose and low PPFD), about 2 to 2.5 times higher. A similar response was observed for root regeneration.

Anthocyanin accumulation and expression pattern of anthocyanin biosynthesis genes were investigated in developing coleoptiles of wheat (Triticum aestivum L.). In epidermal cell layers of the growing coleoptiles of cv. Hope, anthocyanins started to accumulate between day 2 and 3 after germination, reached their maximum on day 6 and then decreased while another cultivar, Chinese Spring (CS) did not accumulate anthocyanin pigments. None of the six anthocyanin biosynthesis genes was upregulated in coleoptiles of both cvs. grown in the dark, whereas all genes were activated by light in coleoptiles of cv. Hope. Transcript levels of all the six genes were relatively low on day 2, increased from days 3 to 5 and then declined to almost non-detectable levels on day 6. In coleoptiles of CS grown in the light, the early biosynthesis genes (EBGs) were expressed, but the three late biosynthesis genes (LBGs) were not.

A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige and Skoog (MS) medium supplemented with 0.025 mg dm^-3 thidiazuron (TDZ) and 3 mg dm^-3 AgNO₃. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm^-3 α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %.

The objective of this study was to determine the development of the antioxidant enzymes induced by drought stress in white clover (Trifolium repens L.) leaves. Water stress was imposed during 28 d by decreasing the daily irrigation. Leaf water potential (Φ_w) gradually decreased from -0.46 to -2.33 MPa. For the first 7 d, dry mass (DM), H₂O₂ and lipid peroxidation were not significantly affected by water deficit. From 14 d of treatment, water stress decreased dry mass and increased content of reactive oxygen species (O₂ ^.- and H₂O₂) and oxidative stress (malondialdehyde content). The ascorbate peroxidase (APOD) was activated most rapidly, already during the first week of water stress, but then its activity slowly decreased. Activation of superoxide dismutase (SOD) and catalase (CAT) by water deficit continued during the 14 d (Φ_w ≥ -1.65 MPa) and then their activities remain on the similar level. The activity of guaiacol-peroxidase (GPOD) increased mostly under progressive water stress and was correlated with increase in lipid peroxidation and growth restriction.

Cuttings from two contrasting Populus cathayana Rehder populations originating from Hanyuan (wet climate) and Ledu (dry climate) in western China were grown in a greenhouse to determine the effects of drought, shade and their interaction on the morphological and physiological traits of leaves. The dry climate population was more droughttolerant than the wet climate population, as indicated by smaller decreases in the leaf relative water content (RWC) and net photosynthetic rate (PN), as well as by greater increases in antioxidative enzyme activities and free proline content under drought. On the other hand, the negative effects of shade on leaf traits were more pronounced in the dry climate population, which suggested that the dry climate population was more light-demanding. In addition, moderate shade alleviated the drought stress of P. cathayana not only by improving the leaf RWC but also by maintaining the positive carbon balance. In contrast, severe shade aggravated drought stress as indicated by a pronounced decrease in leaf size, carbon and nitrogen contents, maximum PN, free proline content and antioxidative enzyme activities.

An attempt has been made to determine if drought-induced proteins could be used as a selection marker to differentiate between tolerant and sensitive cultivars. Three Indian tomato (Lycopersicon esculentum Mill.) cultivars (Pusa Ruby, Arka Vikas and Pusa Early Dwarf) were subjected to drought stress in vivo as well as in vitro and the pattern of polypeptide expression was determined using one-dimensional SDS-PAGE. In all the three cultivars, a new 29 kDa polypeptide accumulated in leaves, in response to gradual drought stress and its accumulation was fastest in Pusa Ruby. Drought stress also resulted in an increase in ion leakage from leaf discs of all the three cultivars but the rate was lower in Pusa Ruby than in other two. Therefore, it was concluded that Pusa Ruby is most tolerant to drought stress among the three tomato cultivars investigated.

To reveal the maternal donor of species in genus Kengyilia, the chloroplast trnL-F sequences of 14 Kengyilia species and several related diploid species were analyzed by using Maximum Parsimony (MP) and Bayesian Inference (BI) methods. The species in Kengyilia were clustered in different clades, which indicated that Agropyron (P) is the likely maternal genome donor to Kengyilia melanthera, K. mutica and K. thoroldiana, while the maternal donor to Kengyilia batalinii, K. nana, K. kokonorica, K. kaschgarica, K. hirsuta, K. alatavica, K. gobicola, K. zhaosuensis, K. rigidula, K. longiglumis and K. grandiglumis was St or Y Roegneria genome.

A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared to the whole plasmid. The Gfp expression was stable up to the T₂ generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation in bentgrass with possible applications to other plant species.

The water-water cycle which may be helpful for dissipating the excitation pressure over electron transport chain and minimizing the risk of photoinhibition and photodamage was investigated in rice after 10-d P-deficient treatment. Net photosynthetic rate decreased under P-deficiency, thus the absorption of photon energy exceeded the energy required for CO₂ assimilation. A more sensitive response of effective quantum yield of photosystem 2 (Φ_PS2) to O₂ concentration was observed in plants that suffered P starvation, indicating that more electrons were transported to O₂ in the P-deficient leaves. The electron transport rate through photosystem 2 (PS 2) (J_f) was stable, and the fraction of electron transport rate required to sustain CO₂ assimilation and photorespiration (J_g/J_f) was significantly decreased accompanied by an increase in the alternative electron transport (J_a/J_f), indicating that a considerable electron amount had been transported to O₂ during the water-water cycle in the P-deficient leaves. However, the fraction of electron transport to photorespiration (J_o/J_f) was also increased in the P-deficient leaves and it was less sensitive than that of water-water cycle. Therefore, water-water cycle could serve as an efficient electron sink. The higher non-photochemical fluorescence quenching (q_N) in the P-deficient leaves depended on O₂ concentration, suggesting that the water-water cycle might also contribute to non-radiative energy dissipation. Hence, the enhanced activity of the water-water cycle is important for protecting photosynthetic apparatus under P-deficiency in rice.

Changes in hydraulic conductivity (K_h) and pH were measured in stem segments of laurel (Laurus nobilis L.) during perfusion with iso-osmotic solutions of KCl, NaCl and sucrose. Sucrose had no effect on K_h while 100 mM NaCl or KCl induced up to 22 and 35 % increase of K_h with respect to deionized water, respectively. Increases in K_h were accompanied by a sharp drop in pH from 6.0 (inlet solution) to 5.0 (outlet solution). The same effect was observed with both KCl and NaCl solutions but not in the case of sucrose. Also, similar changes of K_h and pH were observed for stems killed after immersion in hot water. Our results might provide further evidence for ion-mediated regulation of xylem hydraulic conductivity based on the hydrogel properties of pectins at the pit membrane level.

Shoot and root mass of tobacco plants treated with only 0.05 µM boron was decreased by 25 and 50 %, respectively, when compared to plants sufficiently supplied with B (2 and 5 µM). Leaf B content of 0.05 µM B-treated plants decreased (about 80-90 %) when compared to 2 µM B treated plants; this drop of B content were not as marked (about 25-45 %) in roots. Leaf and root nitrate contents in B-deficient plants were 45-60 % and 35-45 % lower, respectively, than those from 2 and 5 µM B treated plants. It is suggested that B deficiency might decrease nitrate uptake rather than nitrate reductase activity in tobacco plants.

In the present study, we investigated tiller formation in rice cultivated under red radiation (R), red supplemented with 10 % of blue radiation (RB), and white radiation (W). In addition, expression of three genes related to tiller formation, OSH1, MOC1, and OsTB1, was analyzed at both vegetative and reproductive stages. RB promoted the outgrowth of tiller buds and increased tiller numbers significantly. Transcription of MOC1 and OsTB1 in RB was higher than in R, whereas OSH1 expression was independent on radiation quality.

In red clover (Trifolium pratense L.) four unique cDNAs encoding phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were identified (PAL1-4). PAL2-4 encode nearly identical proteins (> 97 %) that are only 89 % identical to that encoded by PAL1. Under normal growing conditions, in young leaves and flowers PAL1 mRNA accumulates to higher levels than that of PAL2-4 whereas in mature leaves and stems, mRNA levels are similar for PAL1 and PAL2-4. Treatment of red clover seedlings with yeast elicitor preparation results in an approximately six-fold induction of PAL2-4 transcripts within 1 h of treatment but only a modest induction of PAL1 transcripts. These results suggest that while both classes of enzymes play a role in biosynthesis of phenylpropanoid compounds under normal growing conditions, PAL2-4 enzymes are also involved in pathogen defense responses.

The effect of esculetin and some related coumarins (coumarin, 7-hydroxycoumarin and scopoletin) on growth of pumpkin (Cucurbita maxima Duch.) seedlings and the activities of pumpkin glutathione S-transferases (GSTs) were investigated. Coumarin and esculetin affected the growth of seedlings. The hypocotyls of affected seedlings became weak and bent at the mid region, roots became very soft with brownish discoloration, and finally seedlings died. Among the compounds tested, only esculetin inhibited CmGSTU3 and CmGSTU2 activities measured with 1-chloro-2,4-dinitrobenzene (CDNB) and at a concentration of 22 µM, it inhibited the activity of CmGSTU3 by 50 %.

The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones, while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones.

The response of Camellia sinensis (L.) O. Kuntze cultivars Chinary and Assamica to Cu and Al stresses was investigated. Exposure to 100 µM CuSO₄ or 100 µM AlCl₃ led to accumulation of reactive oxygen species (ROS) more in Assamica than in Chinary. Proline content was higher in Chinary compared to Assamica, while chlorophyll and protein contents decreased upon Cu and Al exposure in both the cultivars. Expression of glutathione biosynthetic enzymes γ-glutamylcysteinyl synthetase (γ-ECS) and glutathione synthetase (GSHS) was elevated. Phytochelatin synthase (PCS), an enzyme involved in phytochelatins synthesis by using glutathione as a substrate was up-regulated at its transcript level more in Chinary than in Assamica. These results suggest that Chinary could be more tolerant than Assamica.