Early morphogenetic events and repetitive embryogenesis from callus culture of betel nut palm (Areca catechu L.) were studied using scanning electron microscopy. On Murashige and Skoog (MS) medium supplemented with 2 mg dm^-3 dicamba, callus culture has capacity to form plantlets via somatic embryogenesis and to form secondary embryos for about 4 years. However, various abnormal embryos without differentiation of the leaf sheath and shoot apical meristem were observed, which showed bell-shaped and then cup-shaped or mushroom-shaped structures. These abnormal embryos contained distinctive structures, including a disk-shape interior region, surfaces with grooves and a stalk-like posterior region. During subculture, these abnormal embryos enlarged, became deformed and gradually lose their shape and then converted into nodular, compact embryogenic callus. It was also found that secondary embryos originated from interior surfaces or posterior regions of abnormal embryos, and gave rise to the next cycle of normal and abnormal embryos.

In the yeast Saccharomyces cerevisiae, the molecular chaperone HSP26 has the remarkable ability to sense increases in temperature directly and can switch from an inactive to a chaperone-active state. In this report, we analyzed the effect of expression of HSP26 in Arabidopsis thaliana plants and their response to high temperature stress. The hsp26 transgenic plants exhibited stronger growth than wild type plants at 45 °C for 16 h. The chlorophyll content and chlorophyll fluorescence decreased much more in wild type than in transgenic plants. Moreover, the transgenic plants had higher proline and soluble sugar contents, and lower relative electrical conductivity and malondialdehyde contents after high temperature stress. Furthermore, we found that over-expression of HSP26 in Arabidopsis increased the amount of free proline, elevated the expression of proline biosynthetic pathway genes and therefore enhanced Arabidopsis tolerance to heat stress.

Present cytological investigations from the cold desert regions of Lahaul-Spiti and Kinnaur (India) record the first ever tetraploid (2n=32) chromosome count and cytomixis in Clematis orientalis L. var. acutifolia Hook. f. et Thoms. The phenomenon of cytomixis (9.33-29.80 %) involving chromatin transfer among 2-3 proximate pollen mother cells (PMCs) during male meiosis occurs through narrow and broad cytoplasmic channels from early prophase to tetrad stage. However, frequency of its occurrence during the later meiotic stages is rather low. Chromatin transfer results into the formation of hypo-, hyperploid and enucleated PMCs. Various meiotic abnormalities associated with cytomixis such as chromatin stickiness, pycnotic chromatin, interbivalent connections, out of plate bivalents, late disjunction of bivalents, and laggards and bridges resulted into some pollen sterility (16.33-49.30 %) and heterogeneous pollen grains size.

Leaf explants of Jatropha curcas cultured on Murashige and Skoog's (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0-5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.

The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3' truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.

Sorghum bicolor L. Moench cv. SPV462 was transformed with the mtlD gene encoding for mannitol-1-phosphate dehydrogenase from E. coli with an aim to enhance tolerance to water deficit and NaCl stress. Transgene (pCAM mtlD) integration and expression were successfully confirmed by PCR, Southern, RT-PCR and Western analysis. Segregation analysis based on germination of T0 seed on hygromycin-supplemented medium revealed an expected Mendelian ratio 3:1 in lines 5, 72 and 75. Retention of leaf water content was remarkably higher in transgenic leaf segments when exposed to polyethylene glycol 8000 (-2.0 MPa), as compared to the untransformed controls. Another significant finding is that the transgenics maintained a 1.7 to 2.8 fold higher shoot and root growth, respectively, under NaCl stress (200 mM) when compared to untransformed controls. These results demonstrate that engineering mannitol biosynthetic pathway into sorghum can impart enhanced tolerance to water deficit and salinity.

Effect of gibberellic acid (GA₃) combined with saponin on shoot elongation of Asparagus officinalis was evaluated in tissue culture. Addition of saponin to GA₃ supplemented Murashige and Skoog (MS) basal medium showed a dose depended effect on shoot length of Asparagus officinalis. However, increasing concentration of saponin above 3.0 mg dm^-3 decreased shoot length and showed yellowing and thinning of shoots. Saponin (3.0 mg dm^-3) + GA₃ (0.2 mg dm^-3) mixture treated by variable duration of sonication (0, 1, 3, 5, 7, 9, 11, 13 and 15 min) were evaluated for shoot elongation on MS basal medium. The highest shoot lengths (14.4 ± 0.3 and 15.1 ± 0.1 cm) were found after 5 and 7 min sonication.

The aim of this work was to investigate the role of the antioxidant enzymes in salt tolerance comparing the salt-sensitive (Pérola) and a salt-tolerant (Pitiúba) cultivar of cowpea [Vigna unguiculata (L.) Walp.]. Salt stress (100 mM NaCl for 8 d) reduced the leaf growth rate more in the sensitive cultivar. The salt-induced decrease in the relative water content, Na⁺ accumulation and increase in leaf electrolyte leakage was similar in both cultivars. Salt stress induced a higher increase in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and phenol peroxidase (POX) in the tolerant cultivar than in sensitive one.

The Triticum aestivum - Aegilops biuncialis (2n=4x=28; U^bU^bM^bM^b) disomic addition lines 2M^b, 3M^b, 7M^b and 3U^b were crossed with the wheat cv. Chinese Spring ph1b mutant genotype in order to induce homoeologous pairing, with the final goal of introgressing Ae. biuncialis chromatin into cultivated wheat. Wheat-Aegilops homoeologous chromosome pairing was studied in metaphase I of meiosis in the F₁ hybrid lines. Using U and M genomic probes, genomic in situ hybridization (GISH) demonstrated the occurrence of wheat-Aegilops homoeologous pairing in the case of chromosomes 2M^b, 3M^b and 3U^b, but not in the case of 7M^b. The wheat-Aegilops pairing frequency decreased in the following order: 2M^b > 3M^b > 3U^b > 7M^b, which may reflect differences in the wheat-Aegilops homoeologous relationships between the examined Aegilops chromosomes. The selection of wheat-Aegilops homoeologous recombinations could be successful in later generations.

The analyses of some antioxidant enzyme activities were carried out in the course of strawberry fruits development and ripening. The catalase activity was maximum in small-sized green fruits, it decreased in middle-sized green fruits and increased again during the ripening stages. The highest superoxide dismutase and peroxidase activities were observed in white fruits.

An efficient plant regeneration protocol was developed from rhizomes of two Curcuma species C. longa and C. amada. Response was highly dependent on the season, with above 69 % of culture developing adventitious shoots during spring. Greatest regeneration and multiplication was observed in modified Murashige and Skoog (MS) medium supplemented with 13.31 μM benzyladenine and 2.68 μM α-naphthalene acetic acid (NAA) in C. longa or 2.46 μM indolebutyric acid in C. amada. Effect of sugars and agar at different concentrations were also studied and 2 % maltose and 0.7 % agar were found optimum for shoot multiplication and regeneration. Most plantlets developed roots simultaneously but others formed roots when subcultured in 1/2 MS medium supplemented with 2.68 μM NAA. Plants were successfully hardened in greenhouse with 80 % survival. The genetic purity of micropropagated plantlets was analyzed using RAPD and protein profiles.

The activities of antioxidative enzymes and contents of proline and total phenolics were assayed in roots of two maize (Zea mays L.) genotypes grown in a medium containing nitrate (NO₃ ^-) or both nitrogen forms, nitrate and ammonium (NH₄ ⁺/NO₃ ^-). An increase in the activities of class III peroxidases (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), ascorbate oxidase (AO) and proline content, and decrease in phenolic content were observed in NH₄ ⁺/NO₃ ^- in comparison with NO₃ ^- grown plants. When polyethylene glycol (PEG) was added to both nitrogen treatments, the content of total phenolics and proline was increased, especially in NH₄ ⁺/NO₃ ^- treatment. The PEG treatment decreased enzyme activities in NH₄ ⁺/NO₃ ^- grown plants, but in NO₃ ^- grown plants activities of POD and SOD were increased, opposite to decreased APX and AO. Isoelectric focusing demonstrated increased activities of acidic POD isoforms in PEG treated NO₃ ^- grown plants, and lower activities of both, acidic and basic isoforms in NH₄ ⁺/NO₃ ^-grown plants.

Dune reed (DR) and swamp reed (SR) are two ecotypes of reed (Phragmites communis Trin.) that displayed differences in stress tolerance. To uncover the molecular basis for such difference, the effects of heat stress were studied using the calli derived from the two ecotypes. Heat stress caused increased ion leakage, inhibited growth, decreased cell viability, and elevated hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents in the calli of both ecotypes, but DR callus showed better heat tolerance than SR callus. In DR callus, heat stress caused significant increase in the endogenous ABA content but not in SR callus. Application of fluridone (an ABA synthesis inhibitor) aggravated the heat stress damages on the DR callus whereas it had only minimal impact on the SR callus. Exogenous application of ABA alleviated the heat stress symptoms in the calli of both ecotypes. ABA treatment increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase, and also decreased H₂O₂ and MDA contents. These results indicate that the ability of ABA synthesis under heat stress is a key factor attributing to the higher heat tolerance of DR than SR.

This communication examines the role of small heat shock proteins (sHsps) targeted to mitochondria (Mt) and endoplasmic reticulum (ER) in tomato plants (Lycopersicon esculentum Mill.) under heat stress. Genetic response of transgenic and wild type plants varied under optimum, moderately elevated and elevated temperature. In optimum temperature higher biomass was recorded in wild type than the transgenic lines, whereas in moderately elevated temperature biomass increased in Mt-sHsp line. Also, net photosynthetic rate (P_N) increased in Mt-sHsp line in both the elevated temperatures, though higher in moderately elevated. Cell membrane stability (CMS) improved in all the lines after exposure to elevated temperatures, but always remained higher in transgenic lines. Transgenic lines expressed sHsps in different temperature regimes in both vegetative and reproductive parts, while wild type expressed such proteins only after 1 h of heat shock.

Nitric oxide production, nitric oxide synthase (NOS) and mitochondrial nitrite-reducing activities in roots, leaves and stems of different developmental stages were investigated, using potted 3-year-old apple (Malus domestica Borkh.) trees. The arginine-dependent NOS activity is sensitive to NOS inhibitor L-NAME and aminoguanidine (AG), with L-NAME being more effective than AG. Endogenous NO production, NOS and mitochondrial nitrite-reducing activities are predominately presented in young leaves and especially in young white roots and young stems. Root and stem mitochondria can reduce nitrite to nitric oxide at the expense of NADH, however, this mitochondrial nitrite-reducing activity is absent in leaves.

Leaves from 14-d-old Capsicum annuum L. cv. Anaheim seedlings were cultured on Murashige and Skoog (MS) medium containing different combinations of indole-3-acetic acid (IAA) and 6-benzyladenine (BA). After 3 months, cultures were transferred to new medium where BA was replaced with 9 μM isopentenyladenine (2iP) to enhance the growth of shoot buds. Developing shoots were elongated and rooted on MS medium enriched with 9 μM indole-3-butyric acid (IBA). All cultures were maintained in 250 cm³ baby jars covered with a clear polypropylene lid with or without microporous polypropylene membrane. Vessel type and plant growth regulators significantly affected callus morphogenic appearance, organogenesis and in vitro plantlet growth. Ventilated vessels supported photomixotrophic culture and improved regeneration and growth of plantlets. Higher plantlet dry mass and content of photosynthetic pigments, and lower stomatal density of plantlets grown in ventilated than in non-ventilated vessels facilitated ex vitro acclimation and growth.

The effects of three cytokinins, kinetin 4.5 μM (Kin), 6-benzylaminopurine 4.5 μM (BA) and N-phenyl-N'1,2,3- thiadiazol-5-yl-urea 4.5 μM (TDZ), and the effects of different treatment duration on the regeneration of adventitious shoots from quince (Cydonia oblonga Mill.) leaves were studied. In a first experiment, leaves treated with Kin for 0, 8, 16 and 24 d were transferred to BA or TDZ-containing growth medium. In a second experiment TDZ applied for 0, 4, 8, 12, 16 and 24 d was followed by BA. All treatments included 0.5 μM α -naphthaleneacetic acid (NAA). In the sequence Kin-BA, the production of adventitious shoots decreased and reddish-coloured nodular structures (RNS) of meristematic appearance increased with increasing duration of Kin treatment, while somatic embryo formation was optimal at 8 d. In the Kin-TDZ sequence, shoot production was initially pronounced, but it declined with increasing duration of the Kin treatment, while the number of roots, somatic embryos and RNS increased. TDZ-BA treatments induced marked shoot production, which gradually increased with increasing duration of TDZ treatment. The presence of TDZ and a long treatment duration appeared to be very important factors in inducing caulogenesis. Kin appeared to be effective in shoot induction but not in shoot development; the results of this work demonstrate that RNS were adventitious shoots blocked at an early developmental stage on account of insufficient cytokinin activity. BA was less effective than TDZ in inducing shoot regeneration. Finally, both Kin and BA applied after 2,4-D treatment promoted somatic embryo induction.

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2-3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.

The objective of this work was to test whether Ca²⁺, a second messenger in stress response, is involved in ABA-induced antioxidant enzyme activities in Stylosanthes guianensis. Plants were sprayed with abscisic acid (ABA), calcium channel blocker, LaCl₃, calcium chelator, ethylene glycol-bis(β-amino ethyl ether)-N,N,N', N'-tetraacetid acid (EGTA), and ABA in combination with LaCl₃ or EGTA. Their effects on superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and chilling resistance were compared. The results showed that ABA decreased electrolyte leakage and lipid peroxidation but increased maximum photochemical efficiency measured as variable to maximum fluorescence ratio (F_v/F_m) under chilling stress. Treatment with LaCl₃ or EGTA alone and in combination with ABA increased electrolyte leakage and lipid peroxidation, decreased Fv/Fm, suggesting that the block in Ca²⁺ signalling decreased chilling resistance of S. guianensis and the ABA-enhanced chilling resistance. ABA-induced SOD and APX activities were suppressed by LaCl₃ or EGTA. The results suggested that Ca²⁺ is involved in the ABA-enhanced chilling resistance and the ABA-induced SOD and APX activities in S. guianensis.

Plant β-glucosidases catalyze the hydrolysis of glycosidic linkages and play a vital role in defense against pathogens and stress. The present work investigated the relationship between leaf development and β-glucosidase protein content in Olea europea L. (cv. Picual) leaves. The total chlorophyll content increased with leaf age in current-season leaves. Immunoblot analysis revealed that the content of 61 kD protein of β-glucosidase also increased with leaf age, and that the enzyme existed in three isoforms (pI 5.8-6.2). Statistical analysis indicated a strong correlation between chlorophyll and β-glucosidase protein contents.

Recent developments in knowledge about the interactions between lichens and heavy metals at different levels, from populations to cells and from ecology to molecular biology are reviewed. Sources of heavy metals, mechanisms of heavy metal accumulation and detoxification by lichens are discussed. Special emphasis is placed on ultrastructural changes as well as physiological parameters such as membrane integrity, pigment composition, chlorophyll a fluorescence, photosynthesis, respiration, contents of ATP, amino acids, ergosterol, ethylene, non-protein thiols, activity of antioxidant enzymes and expression of stress proteins.

Barley seedlings were pre-treated with 1 and 5 µM H₂O₂ for 2 d and then supplied with water or 150 mM NaCl for 4 and 7 d. Exogenous H₂O₂ alone had no effect on the proline, malondialdehyde (MDA) and H₂O₂ contents, decreased catalase (CAT) activity and had no effect on peroxidase (POX) activity. Three new superoxide dismutase (SOD) isoenzymes appeared in the leaves as a result of 1 µM H₂O₂ treatment. NaCl enhanced CAT and POX activity. SOD activity and isoenzyme patterns were changed due to H₂O₂ pre-treatment, NaCl stress and leaf ageing. In pre-treated seedlings the rate of ¹⁴CO₂ fixation was higher and MDA, H₂O₂ and proline contents were lower in comparison to the seedlings subjected directly to NaCl stress. Cl^- content in the leaves 4 and 7 d after NaCl supply increased considerably, but less in pre-treated plants. It was suggested that H₂O₂ metabolism is involved as a signal in the processes of barley salt tolerance.

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.

An experiment was conducted with two contrasting pigeon pea (Cajanus cajan L.) genotypes, ICPL 84023 (tolerant) and ICP 7035 (susceptible), to study the physiological and molecular basis of waterlogging tolerance in relation to oxidative stress and antioxidant enzyme activities. Waterlogging resulted in visible yellowing and premature senescence of leaves, and greater decline in relative water content, chlorophyll content, and membrane stability index in ICP 7035 than in ICPL 84023. Superoxide radical and hydrogen peroxide contents increased at day 4 and 6 of waterlogging probably due to activation of NADPH-oxidase. O₂ ^.- production was inhibited, by diphenylene iodonium chloride, a specific inhibitor of NADPH oxidase and expression of NADPH oxidase-mRNA was increased under waterlogging condition in ICPL 84023. ICP 7035 showed higher contents of ROS in control condition and after recovery, however, during waterlogging the O₂ ^.- production was higher in ICPL 84023. Activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase and catalase increased under waterlogging more in ICPL 84023 than in ICP 7035. Cu/Zn-SOD and APX-mRNA expression in 24-h waterlogged plants showed enhanced expression in ICPL 84023 compared to ICP 7035. The cloning and sequencing of APX gene of tolerant and susceptible genotypes yielded cDNAs of 622 and 623 bp, having 95 % homology with each other and 92 % with the corresponding sequences of Vigna unguiculate APX-gene.

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.

Thalli of Xanthoparmelia somloensis with natural content of polyols (control) and polyol-free thalli (acetone-rinsed) were used to study ribitol effects at low temperatures. Thalli segments were cultivated in ribitol concentration of 32 or 50 mM for 168 h at temperatures +5, 0, and -5 °C. The chlorophyll fluorescence parameters (potential yield of photochemical reactions in PS 2 (variable to maximum fluorescence ratio, F_v/F_m), effective quantum yield of photochemical reactions in PS 2 (Φ_PS2), and non-photochemical quenching (NPQ) were monitored in 24-h intervals using an imaging system. The effect of 32 mM ribitol on F_v/F_m and Φ_PS2 was apparent only at -5 °C, however, the effect was seen throughout the whole exposure. Surprisingly, 50 mM ribitol concentration treatment led to a decrease in F_v/F_m and Φ_PS2 and to an increase in NPQ values at -5 °C, while no change was observed at 0 °C and +5 °C. Acetone-rinsing caused decrease of F_v/F_m, Φ_PS2 and NPQ.

To get deeper insight on the molecular mechanism underlying production of volatile compounds in apple (Malus domestica Borkh.), we performed the isolation and expression analysis of one R2R3-type MYB gene named MdMYBB. The amino acid sequence and the structural features of MdMYBB highly resembled those of PhODO1, which is a key regulator for floral scent biosynthesis in petunia. The expression of MdMYBB was repressed concomitantly with the inhibition of ethylene production, which regulates the volatile synthesis in apple. However, MdMYBB expression was not detected in the flesh from nearly ripened apple fruits, although the detection of exogenous volatiles had actually occurred in the same portion. In addition, overexpression of MdMYBB did not cause any induction of the volatile compounds in the transgenic tobacco lines. Thus, the features of MdMYBB were not in accordance with the aroma volatile emission, unlike the case of PhODO1, suggesting that MdMYBB may not be involved in the regulation of the biosynthesis for apple aroma volatiles. On the basis of the specific expression patterns, we discussed possible physiological roles of MdMYBB in apple.

Non-transformed and transformed embryogenic cultures of alfalfa (Medicago sativa L. cv. Zaječarska 83), long-term maintained on growth regulator-free medium, were histologically analyzed. In all examined cultures, somatic embryos at various stages of development were observed and secondary embryos were formed in the cotyledonary, hypocotylary and radicular region of the primary embryos. Detailed histological analysis of the torpedo shape somatic embryo revealed that secondary somatic embryos arose directly from single epidermal cells of hypocotylary axis after an unequal periclinal division. Bipolar proembryos were composed of one smaller cytoplasm rich cell and one larger more vacuolated cell. Further cell division pattern was similar for both non-transformed and transformed embryos. However, multicellular origin of secondary embryos in a direct process and even from callus can not be excluded.