Biochemical and molecular markers have been used on eleven species of Cucurbitaceae collected from lower Gangetic plains. Six enzyme systems were selected. Among 40 primers examined, 14 random amplified polymorphic DNA (RAPD) and 10 inter-simple sequence repeat (ISSR) primers were selected for the analysis. Generated RAPD (100) and ISSR (100) fragments showed high variations among the species. Jaccard similarity coefficients were used for the evaluation of pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate interspecific diversity. Further, principal coordinate analysis was performed to evaluate the resolving power of the three marker systems to differenciate among the species.

Unfavourable environment brings many kinds of stresses to plants. To survive such stresses, efficient resistance is required for the plants. Multifunctional genes enable the cross-talk among the various abiotic stress resistance systems. This paper reviews the action mechanisms of multifunctional genes. These genes can be classified into three groups: genes encoding diverse proteins through mRNA splicing (e.g. AOX in rice); genes like BADH, P5CS and HAV that control drought, salinity, osmotic and heat stress resistance; and a gene family, for example AQP, controlling transport of many compounds including water and nutrients. These genes participate in signal sensing and transduction, transcriptional regulation and functional gene activation during stress resistance induction. Furthermore, it should be noted that, under abiotic stresses, the regulation cascades are mutually interdependent and there also exists a close correlation between those cascades and normal plant growth and development.

Plantlet regeneration through indirect somatic embryogenesis was attempted from rhizome derived callus of Cymbopogon winterianus Jowitt (cv. Jorlab2). Optimum callus was induced on Murashige and Skoog (MS) basal medium supplemented with 4 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D). Initially the callus was friable, shiny white and watery in nature. After subculturing on MS medium containing 2,4-D and kinetin (Kn), callus was transferred onto the MS medium supplemented with 2,4 -D, Kn and coconut water to induce somatic embryogenesis. Optimum somatic embryogenesis (78.33 %) was achieved on MS medium containing 3.0 mg dm^-3 2,4-D and 0.5 mg dm^-3 Kn. High frequency (65 %) plantlet conversion from embryos was achieved in MS medium supplemented with 2 mg dm^-3 N⁶-benzyladenine (BA), 0.5 mg dm^-3 Kn, 0.2 mg dm^-3 calcium pantothenate and 0.2 mg dm^-3 biotin.

Chlorophyll (Chl) degradation was found to be related to the endogenous gibberellin (GA) content in shoots during senescence in the perennial plant Paris polyphylla var. yunnanensis (Franch.). Treatment with gibberellic acid (GA₃) significantly increased the content of endogenous GAs (GA₄ + GA₇), retarded the senescence of shoots, and the degradation of proteins and Chl. Chlorophyllase, Mg-dechelation and peroxidase activities increased more in control plants than in those treated with GA₃. GA₃ treatment also protected lipoxygenase activity, which decreased significantly in control plants.

In previous experiments elevated but sub-symptomatic applications of Zn (0.1 mM and 1 mM) caused impairments in growth parameters and photosynthetic performance of Populus × euramericana (Dode) Guinier clone I-214. The aim of this work was to evaluate leaf morphological and anatomical traits in this clone in response to the same Zn concentrations. The results showed that Zn treatments induced variations in leaf dry mass, area, mesophyll thickness, intercellular spaces, stomatal density and size. Stronger modifications, especially concerning stomata characteristics induced by 1 mM Zn, were consistent with physiological impairments while those induced by 0.1 mM Zn suggested a compensatory strategy for maintaining functional integrity.

Expressed sequenced tags containing simple sequence repeats (EST-SSRs) were used to identify molecular markers associated with yellow rust resistance in wheat (Triticum aestivum L.). A cross between yellow rust resistant (PI178383) and susceptible (Harmankaya99) wheat genotypes was performed and respective DNA pools from the resistant and susceptible F₂ seedlings were constructed. 78 EST-SSR primers were used for bulked segregant analysis and one EST-SSR marker (Pk54), identified as 200 bp fragment, was present in the resistant parent and resistant F₂ hybrids but not in the susceptible ones. 108 wheat genotypes differing in yellow rust resistance were screened with Pk54 and 68 % of the wheat genotypes, known to be yellow rust resistant, had the Pk54 marker, further suggesting that the presence of this marker correlates with yellow rust resistance.

The effect of heterosis on callus induction, callus mass and number of regenerated plants from mature embryo cultures of winter durum wheat (Triticum durum Desf.) hybrids was studied. A total of 14 F₁ hybrids and their parents were used for mature embryo culture. The statistical analysis of the results revealed that positive heterosis in callus mass was determined in only one F₁ hybrid and in number of regenerated plants in two F₁ hybrids. Plants regenerated in vitro were successfully established in soil. Hybrid genotypes may be used to obtain callus and regenerated plants with vigour comparable to their parents.

5-Methyltryptophan (5MT), a tryptophan analog, resistant M4 rice mutants with high free amino acid contents were obtained through in vitro mutagenesis. To evaluate the 5MT resistance mechanism, a cDNA library was constructed by using the leaves and roots of the 5MT resistant mutant plants. Expressed sequenced tags (ESTs) of 1 019 randomly selected clones were analyzed and then assembled 588 unigens. A total of 389 unigenes had significant homologies with known protein sequences at the NCBI database and the remaining 199 unigenes were designated unidentified genes. These unigens were grouped into 13 categories according to their putative functions. Of the 233 randomly selected clones, 25 were identified as differentially expressed genes between 5MT resistant and 5MT sensitive wild type plants. For further study of the differential expression of the genes, expression patterns of 12 genes related to various biological functions were evaluated in response to 5MT treatment in both the resistant plants and sensitive plants. All of the tested 12 genes exhibited higher expression levels in mutant plants than wild type plants under the 5MT inhibition. These expression patterns of the 12 genes suggested that the genes related to 5MT resistance in the rice mutants have a variety of functions, and yield remarkably diverse expression patterns upon 5MT treatment. Many genes that were identified tend to be related to defense and stress responses, suggesting "cross-talking" between biotic/abiotic stresses including the 5MT treatment. Therefore, 5MT resistant mutants might be of value for identifying genes related to plant defenses and stresses.

The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied. The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots was similar in both types of plants.

Differences of both amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) polymorphisms were compared between the 60-d-old rice (Oryza sativa L. cv. DH7) and F3 rice plants (SP3) derived from seed, which endured a 7-d-space flight in March 2002. Total leaf AFLP DNA bands amplified from 22 primer pairs were 537 in DH7, whereas 562 in SP3. From the total 267 SSR DNA bands generated by 267 primer pairs, 39 were polymorphic with 22 larger (56 %) or 17 smaller (44 %) fragment size bands. The greatest numbers of AFLP DNA bands were amplified by primer E1M1 in DH7 (33) and E3M1 in SP3 (35), whilst the least by E4M3 in DH7 (14) and E5M2 in SP3 (16).

We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.

With improved staining and chromosome preparation techniques, meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetraploid cucumber (Cucumis sativus L.) was studied to understand the correlation between chromosomes behaviour and fertility. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalents were observed in most PMCs at metaphase I. Lagging chromosomes were frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyads, triads, tetrads with micronuclei and polyads were observed at tetrad stage, and the frequencies of normal tetrad with four microcytes were only 55.4 %. The frequency of abnormal behaviour in each stage of meiosis was counted, and the average value was 37.2 %. The normal meiotic process could be accomplished to form the microspore tetrads via simultaneous cytokinesis. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages: single-nucleus early stage, single-nucleus late stage and 2-celled stage. The frequency of abnormalities was low during the process of male gametophyte development. The germination rate of pollen grains was 46.9 %. These results suggested that abnormal meiosis in PMCs was the reason for low pollen fertility in the autotetraploid cucumber.

We tried to determine the mechanisms by which Ca²⁺ mediated NO-induced programmed cell death (PCD) in tobacco protoplasts. Treatment of tobacco protoplasts with the NO donor sodium nitroprusside (SNP) resulted in a rapid [Ca²⁺]_cyt accumulation and decrease in mitochondrial membrane potential (ΔΨ_m) before the appearance of PCD. NO-induced PCD could be largely prevented not only by NO scavenger c-PTIO, but also by EGTA (Ca²⁺ chelator), LaCl₃ (Ca²⁺-channel blocker) or CsA (a specific mitochondrial permeability transition pore inhibitor, which also inhibit Ca²⁺ cycling by mitochondria). All results suggested that NO-induced PCD is mediated through mitochondrial pathway and regulated by Ca²⁺.

The effects of fluoride on the photosynthetic electron transport chain have been studied in spinach thylakoid membranes. Inhibition in photosystem (PS) 2 electron transport rates and a subsequent increase in PS 1 electron transport rate indicated a possibility of state transitions being a mechanism of fluoride action. This hypothesis was further confirmed by the increase in fluorescence emission F_735/685 at 77 K, a decrease in variable to maximum fluorescence ratio (F_v/F_m) at room temperature and increase in the absorption cross section of PS 1 suggesting that fluoride affects distribution of the excitation energy in favour of PS 1 at the expense of PS 2.

Effects of H₂S on seed germination under chromium (Cr) stress were investigated in wheat (Triticum aestivum L.). Under Cr stress, the percentage of germination of wheat seeds decreased, but this decrease could be alleviated by pretreatment with NaHS, an H₂S donor, in a dose-dependent manner. Furthermore, NaHS significantly enhanced the activities of amylase, esterase, superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidase in Cr-stressed germinating seeds, whereas reduced the Cr-induced increase in lipoxygenase activity and over-production of malondialdehyde (MDA) and H₂O₂, and sustained slightly higher content of endogenous H₂S.

Characteristics of chlorophyll fluorescence and antioxidative system were investigated in rice (Oryza sativa L.) super-hybrid Liangyoupeijiu (LYPJ), maternal cultivar Peiai64s, and paternal cultivar indica rice 9311 under chilling stress. During 6-d chilling treatment, chlorophyll content of all three genotypes was gradually declined. However, the decrease in photosystem 2 (PS 2) maximum photochemical efficiency (F_v/F_m) and quantum yield of PS 2 (Φ_PS2) was less expressive in LYPJ than in parental cultivars The electrolyte leakage and malondialdehyde content in all cultivars increased after chilling treatment, but LYPJ exhibited the least increasing tendency. Activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR) were higher in LYPJ than in parental cultivars. The results demonstrated that tolerance to chilling stress in LYPJ might be adopted mostly from its maternal cultivar.

MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pNPI132, was used to produce morphologically normal transgenic Petunia hybrida 'Dainty Lady' employing isopentenyl transferase (ipt) gene as the selection marker gene. β-glucuronidase (GUS) gene was used as model gene of interest. Infected explants were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGR) and antibiotics. Shoots showing extreme shooty phenotype (ESP) were produced from the adventitious shoots separated from the explants. Visual selection was carried out until production of morphologically normal shoots (approximately 4 months after infection). Histochemical GUS assay detected GUS gene in both ESP and normal shoots. PCR analysis confirmed the presence of model gene (GUS gene) and excision of the selection marker (ipt) gene in the normal transgenic plants. The insertion sites (1-3 for ipt gene and 1-2 for GUS gene) were detected by Southern blot analysis using DIG-labeled probes of both genes. These results show that ipt-type MAT vector can be used successfully to produce marker-free transgenic Petunia hybrida plants on PGR- and antibiotic-free MS medium.

In this study, we wanted to inspect whether the evolutionary driven differences in primary sequences could correlate, and thus predict the genetic diversity of related marker loci, which is an important criterion to assess the quality of any DNA marker. We adopted new approach of quantitative symbolic DNA sequence analysis called DNA random walk representation to study multiallelic marker loci from Begonia × tuberhybrida Voss. We described significant correlation of random walk-derived digital invariants to genetic diversity of the marker loci. Specifically, on the 3D-contour plot of multivariate principal component analysis (PCA), we revealed statistical correlation between the first two PCA factors and the number of alleles per marker locus. Based on that correlation, we suggest that DNA walk representation may predict allele-rich loci solely from their primary sequences, which improves current design of new DNA germplasm identificators.

Suspension cultures of Habanero pepper (Capsicum chinense Jacq.) were exposed to salicylic acid or methyl jasmonate to change secondary metabolism. Both treatments led to the accumulation of capsaicinoids and their late biosynthetic intermediate, vanillin. Both elicitors had a positive effect on the activities of phenylalanine ammonia lyase and coumarate O-methyltransferase, but none of them represented the main limiting step for capsaicinoid accumulation since vanillin contents were two orders of magnitude higher than those of capsaicinoids.

Morphological and molecular characteristics were studied in six wild species of Passiflora. There were statistically significant differences among these six species for all characteristics studied. Intra-specific variability was observed for number of flowers, number of fruits, number of seeds, fruit length, fruit width and leaf area. Cluster analysis using morphological data showed three groups: 1) P. palmeri var. sublanceolata, P. morifolia and P. foetida var. foetida, 2) P. coriacea and P. micropetala, and 3) P. suberosa. The dendrogram constructed using randomly amplified polymorphic DNA (RAPD) data showed six different groups for each species. The genetic distances among the 24 accessions ranged from 0.05 (between P. morifolia accessions P1 and P3) to 0.95 (P. coriacea accession 31 and P. palmeri var. sublanceolata accession 49). The species showed high morphological and molecular inter- and intraspecific variability.

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.

Embryogenic cell suspension cultures of Santalum album were transformed with Agrobacterium tumefaciens harboring pD35SHER plant expression vector having hepatitis B small surface antigen (HBsAg) with a C-terminal ER retention signal. The transformed colonies were selected on culture medium supplemented with kanamycin and subsequently the transgenic nature of these colonies was confirmed by PCR analysis. The expression of HBsAg was confirmed by RT-PCR analysis and Western blot analysis and the expression was quantified using monoclonal antibody-based ELISA. Cell suspension cultures were initiated from the colony with expression of 11.09 μg(HBsAg) g^-1(f.m.). To further increase the expression of HBsAg, transgenic S. album suspensions were cultured on media with various medium additives and cells growing in medium with 30 mM trehalose showed the expression of 19.95 μg(HBsAg) g^-1(f.m.).

Zephyr lily (Zephyranthes grandiflora), an important ornamental plant has been micropropagated in vitro after controlling microbial contamination by a pretreatment with 0.2 % Bavistin and 0.1 % Pantomycin for 4 h before final sterilization with 0.1 % mercuric chloride. In 67 % of the sterile cultures, 11 shoots on average were regenerated directly from basal half of bulb scales in Murashige and Skoog (MS) medium containing 3 % sucrose and 2 mg dm^-3 benzylaminopurine (BAP). Shoots emerged in bunches on a basal achlorophyllous bulbous part. Combination of 2 mg dm^-3 BAP with 1 mg dm^-3 gibberellic acid (GA₃) enhanced shoot growth. Stout roots (maximum of 5-6 per shoot) were developed in presence of 1 mg dm^-3 indole-3-butyric acid (IBA). Micro-bulbs showed potential of regeneration and could be used as secondary explants. The morphologically identical plants derived by in vitro propagation were genetically identical as shown by PCR based ISSR marker analysis of genomic DNA.

We isolated a gene encoding for farnesyl diphosphate synthase (FPS) from Panax ginseng, a species that produces a large quantity of triterpene saponins such as ginsenosides. The deduced amino acid sequence of PgFPS was 77, 84 and 95 % identical to those of Arabidopsis, Hevea, and Centella. Southern blot analysis indicated that P. ginseng contained more than two genes encoding for FPS. When the cDNA of PgFPS was expressed in Escherichia coli, the recombinant enzyme, purified with a His-tag column, was found to possess FPS activity. When cultures of ginseng hairy root were treated with 0.1 mM methyl jasmonate (MJ), PgFPS mRNA was detected within 12 h of the treatment, and achieved maximum after 24 h. Also FPS activity in the hairy root cultures after 12 h of MJ treatment was higher than that of the control.

The transformation of potato (Solanum tuberosum L. cv. Désirée) was extended by the Agrobacterium-mediated biolistic method. Using this approach transgenic shoots could be obtained at a similar frequency to that achieved through conventional biolistics. Leaves from shoot cultures were bombarded with gold particles coated in Agrobacterium tumefaciens cells harboring a binary plasmid encoding three genes of interest in the T-DNA. Nine shoots were obtained from 20 shots, with selection of transgenic shoots on a series of media containing progressively increasing concentrations of hygromycin from 5 to 20 mg dm^-3.

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm^-3 zeatin, 0.1 mg dm^-3 indoleacetic acid and 10 g dm^-3 sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0-2.0 mg dm^-3 gibberellic acid and 4.0-8.0 mg dm^-3 AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.

Content of polyamines and activities of antioxidative enzymes in response to stripe rust disease caused by Puccinia striiformis have been studied in two wheat (Triticum aestivum L.) cultivars PBW 343 (resistant) and HD 2329 (susceptible). Various infection stages ranging from traces to 100 % were collected. Infection leads to stimulation of peroxidase (POD), superoxide dismutase (SOD), catalase, diamine oxidase and polyamine oxidase activities along with increase in putrescine, spermidine and spermine content while ascorbate, tocopherol and chlorophyll content decreased in HD 2329 and no visible symptoms appeared in PBW 343. Histochemical localization pattern of POD and SOD activities revealed correlation with lignin deposition in cell walls.

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm^-3 1-naphthaleneacetic acid (NAA) + 1.0 mg dm^-3 thidiazuron and 1.0 mg dm^-3 NAA + 2.0 mg dm^-3 kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm^-3 NAA + 1.0 mg dm^-3 benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.

An important aspect of heat-shock response of lupin (Lupinus albus cv. Rio Maior) is the formation of cytoplasmic granular aggregates, called heat-shock granules (HSGs). In this study, two-dimensional electrophoresis (2-DE) was used to detect the component proteins of HSG complexes formed in vivo. Evaluation of 2-DE revealed differential expression of several proteins under heat shock conditions when compared with control. Among them, small heat-shock proteins (sHSPs) of 15 to 30 kDa were found to be the major representative proteins along with other proteins of relative molecular mass ranging from 36 to 45 kDa and above.